Spiro-lactam NMDA receptor modulators and uses thereof

ABSTRACT

Disclosed are compounds having enhanced potency in the modulation of NMDA receptor activity. Such compounds are contemplated for use in the treatment of conditions such as depression and related disorders. Orally available formulations and other pharmaceutically acceptable delivery forms of the compounds, including intravenous formulations, are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application under 35 U.S.C. §120 of U.S. patent application Ser. No. 14/764,411, filed on Jul. 29,2015, which application is a U.S. national stage application under 35U.S.C. § 371 of International Application No. PCT/US2014/013623, filedon Jan. 29, 2014, which claims the benefit of and priority to U.S.Provisional Patent Application No. 61/757,934, filed on Jan. 29, 2013,each of which is incorporated by reference herein in its entirety.

BACKGROUND

An N-methyl-d-aspartate(NMDA) receptor is a postsynaptic, ionotropicreceptor that is responsive to, inter alia, the excitatory amino acidsglutamate and glycine and the synthetic compound NMDA. The NMDA receptorcontrols the flow of both divalent and monovalent ions into thepostsynaptic neural cell through a receptor associated channel (Fosteret al., Nature 1987, 329:395-396; Mayer et al., Trends in Pharmacol.Sci. 1990, 11:254-260). The NMDA receptor has been implicated duringdevelopment in specifying neuronal architecture and synapticconnectivity, and may be involved in experience-dependent synapticmodifications. In addition, NMDA receptors are also thought to beinvolved in long term potentiation and central nervous system disorders.

The NMDA receptor plays a major role in the synaptic plasticity thatunderlies many higher cognitive functions, such as memory acquisition,retention and learning, as well as in certain cognitive pathways and inthe perception of pain (Collingridge et al., The NMDA Receptor, OxfordUniversity Press, 1994). In addition, certain properties of NMDAreceptors suggest that they may be involved in theinformation-processing in the brain that underlies consciousness itself.

The NMDA receptor has drawn particular interest since it appears to beinvolved in a broad spectrum of CNS disorders. For instance, duringbrain ischemia caused by stroke or traumatic injury, excessive amountsof the excitatory amino acid glutamate are released from damaged oroxygen deprived neurons. This excess glutamate binds to the NMDAreceptors which opens their ligand-gated ion channels; in turn thecalcium influx produces a high level of intracellular calcium whichactivates a biochemical cascade resulting in protein degradation andcell death. This phenomenon, known as excitotoxicity, is also thought tobe responsible for the neurological damage associated with otherdisorders ranging from hypoglycemia and cardiac arrest to epilepsy. Inaddition, there are preliminary reports indicating similar involvementin the chronic neurodegeneration of Huntington's, Parkinson's, andAlzheimer's diseases. Activation of the NMDA receptor has been shown tobe responsible for post-stroke convulsions, and, in certain models ofepilepsy, activation of the NMDA receptor has been shown to be necessaryfor the generation of seizures. Neuropsychiatric involvement of the NMDAreceptor has also been recognized since blockage of the NMDA receptorCa⁺⁺ channel by the animal anesthetic PCP (phencyclidine) produces apsychotic state in humans similar to schizophrenia (reviewed in Johnson,K. and Jones, S., 1990). Further, NMDA receptors have also beenimplicated in certain types of spatial learning.

The NMDA receptor is believed to consist of several protein chainsembedded in the postsynaptic membrane. The first two types of subunitsdiscovered so far form a large extracellular region, which probablycontains most of the allosteric binding sites, several transmembraneregions looped and folded so as to form a pore or channel, which ispermeable to Ca⁺⁻, and a carboxyl terminal region. The opening andclosing of the channel is regulated by the binding of various ligands todomains (allosteric sites) of the protein residing on the extracellularsurface. The binding of the ligands is thought to affect aconformational change in the overall structure of the protein which isultimately reflected in the channel opening, partially opening,partially closing, or closing.

NMDA receptor compounds may exert dual (agonist/antagonist) effect onthe NMDA receptor through the allosteric sites. These compounds aretypically termed “partial agonists”. In the presence of the principalsite ligand, a partial agonist will displace some of the ligand and thusdecrease Ca⁺⁺ flow through the receptor. In the absence of or loweredlevel of the principal site ligand, the partial agonist acts to increaseCa⁺⁺ flow through the receptor channel.

A need continues to exist in the art for novel and more specific/potentcompounds that are capable of binding the glycine binding site of NMDAreceptors, and provide pharmaceutical benefits. In addition, a needcontinues to exist in the medical arts for orally deliverable forms ofsuch compounds.

SUMMARY

Provided herein, at least in part, are compounds that are NMDAmodulators, for example, partial agonists of NMDA. For example,disclosed herein are compounds represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆alkyl, —OH,        C₁-C₆alkoxy, —OC(O)—C₁-C₆ alkyl and —OC(O)-phenyl (optionally        substituted by one, two or three substituents selected from the        group consisting of halogen, hydroxyl, C₁-C₆alkyl, and        C₁-C₆alkoxy);    -   R₄ is H or C₁-C₆ alkyl; and    -   X is selected from the group consisting of hydrogen,        —C₁₋₆alkylene-C₃₋₆cycloalkyl; C₁₋₆alkylene-heterocycle        (optionally substituted by one, two or three substituents        selected from the group consisting of halogen, hydroxyl,        C₁-C₆alkyl, and C₁-C₆alkoxy), and —C₁₋₆alkylene-heteroaryl        (optionally substituted by one, two or three substituents        selected from the group consisting of halogen, hydroxyl,        C₁-C₆alkyl, and C₁-C₆alkoxy);        or in other embodiments, the variables set forth in formula (I)        are as defined as follows:    -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl (e.g., H);    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆alkyl, —OH,        C₁-C₆alkoxy, —OC(O)—C₁-C₆ alkyl and OC(O)-phenyl (optionally        substituted by one, two or three substituents independently        selected from the group consisting of halogen, hydroxyl,        C₁-C₆alkyl, and C₁-C₆alkoxy);    -   R₄ is H or C₁-C₆ alkyl;    -   X is selected from the group consisting of:        -   (i) hydrogen;        -   (ii) —C₁₋₆alkylene-C₃₋₆cycloalkyl;        -   (iii) —C₁₋₆alkylene-heterocyclyl including from 3 to 6 ring            atoms wherein 1, 2, or 3 of the ring atoms are independently            selected from the group consisting of N, NH, N(C₁-C₃ alkyl),            O, and S; wherein the heterocyclyl is optionally substituted            by one, two or three substituents independently selected            from the group consisting of halogen, hydroxyl, C₁-C₆alkyl,            and C₁-C₆alkoxy);        -   (iv) —C₁₋₆alkylene-C(O)-heterocyclyl including from 3 to 6            ring atoms wherein 1, 2, or 3 of the ring atoms are            independently selected from the group consisting of N, NH,            N(C₁-C₃ alkyl), O, and S; wherein the heterocyclyl is            optionally substituted by one, two or three substituents            independently selected from the group consisting of halogen,            hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy);        -   (v) —C₁₋₆alkylene-heteroaryl including from 5 to 6 ring            atoms wherein 1, 2, or 3 of the ring atoms are independently            selected from the group consisting of N, NH, N(C₁-C₃ alkyl),            O, and S; wherein the heteroaryl is optionally substituted            by one, two or three substituents independently selected            from the group consisting of halogen, hydroxyl, C₁-C₆alkyl,            and C₁-C₆alkoxy;        -   (vi) branched unsubstituted C₃-C₆ alkyl; and        -   (vii) branched C₃-C₆ alkyl substituted with —C(O)NH₂ on one            carbon and —OH on another carbon;    -   and wherein the —NH₂ group attached to the carbon adjacent to        —CH(R₃)(R₄) is optionally substituted with a substituent        selected from —C(O)OR₃₁ and —C(O) R₃₂, wherein:    -   R₃₁ is selected from the group consisting of: C₁-C₆ alkyl; C₁-C₆        haloalkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; C₃-C₁₀ cycloalkyl,        wherein the C₃-C₁₀ cycloalkyl is optionally substituted with        from 1-3 independently selected C₁-C₃ alkyl; —CH₂-C₃-C₁₀        cycloalkyl wherein the C₃-C₁₀ cycloalkyl is optionally        substituted with from 1-3 independently selected C₁-C₃ alkyl;        —CH₂-phenyl, wherein the phenyl is optionally substituted with        from 1-2 substituents independently selected from C₁-C₃ alkyl;        C₁-C₃ haloalkyl; C₁-C₃ alkoxy; C₁-C₃ haloalkoxy; nitro; halo;        SO₂Me, cyano; and —OC(O)CH₃; and —CH₂-pyridyl; and    -   R₃₂ is selected from the group consisting of: H; C₁-C₆ alkyl;        C₁-C₆ haloalkyl; phenyl, wherein the phenyl is optionally        substituted with from 1-2 substituents independently selected        from C₁-C₃ alkyl; C₁-C₃ haloalkyl; C₁-C₃ alkoxy; C₁-C₃        haloalkoxy; nitro; halo; SO₂Me, cyano; and —OC(O)CH₃; and        pyridyl.

In another aspect, disclosed herein are compounds represented by theformula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   R₁ is H or C₁-C₆ alkyl;-   R₂ is H or C₁-C₆ alkyl;-   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, —OH,    C₁-C₆ alkoxy, —OC(O)—C₁-C₆ alkyl and —OC(O)-phenyl;-   R₄ is H or C₁-C₆ alkyl; and-   X is —C₁-C₆alkylene-X′, wherein X′ is a 4- to 6-membered heteroaryl    having 1, 2, or 3 heteroatoms selected from O, N, or S, where X′ is    optionally substituted on a free carbon by one, two or three    substituents selected from the group consisting of halogen,    hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy.

Also provided herein are pharmaceutically acceptable compositionscomprising a disclosed compound, and a pharmaceutically acceptableexcipient. For example, such compositions may be suitable for oral orintravenous administration to a patient.

In another aspect, a method of treating a condition selected from thegroup consisting of autism, anxiety, depression, bipolar disorder,attention deficit disorder, attention deficit hyperactivity disorder(ADHD), schizophrenia, a psychotic disorder, a psychotic symptom, socialwithdrawal, obsessive-compulsive disorder, phobia, post-traumatic stresssyndrome, a behavior disorder, an impulse control disorder, a substanceabuse disorder, a sleep disorder, a memory disorder, a learningdisorder, urinary incontinence, multiple system atrophy, progressivesupra-nuclear palsy, Friedrich's ataxia, Down's syndrome, fragile Xsyndrome, tuberous sclerosis, olivio-ponto-cerebellar atrophy, cerebralpalsy, drug-induced optic neuritis, ischemic retinopathy, diabeticretinopathy, glaucoma, dementia, AIDS dementia, Alzheimer's disease,Huntington's chorea, spasticity, myoclonus, muscle spasm, Tourette'ssyndrome, epilepsy, cerebral ischemia, stroke, a brain tumor, traumaticbrain injury, cardiac arrest, myelopathy, spinal cord injury, peripheralneuropathy, acute neuropathic pain, and chronic neuropathic, in apatient in need thereof is provided. Such methods may compriseadministering to the patient a pharmaceutically effective amount of adisclosed compound or pharmaceutically acceptable salts, stereoisomers,N-oxides, and hydrates thereof

In some embodiments, a contemplated method includes treating depression.For example, depression may include one or more of major depressivedisorder, dysthymic disorder, psychotic depression, postpartumdepression, seasonal affective disorder, bipolar disorder, mooddisorder, or depression caused by a chronic medical condition. In otherembodiments, a contemplated method may treat schizophrenia. Suchschizophrenia may be, for example, paranoid type schizophrenia,disorganized type schizophrenia, catatonic type schizophrenia,undifferentiated type schizophrenia, residual type schizophrenia,post-schizophrenic depression, or simple schizophrenia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the potentiation of [³H]MK-801 binding in the presence ofCompound X.

DETAILED DESCRIPTION

This disclosure is generally directed to compounds that are capable ofmodulating NMDA, e.g., NMDA antagonists or partial agonists, andcompositions and/or methods of using the disclosed compounds.

Definitions

“Treating” includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder and the like.

The term “alkenyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon double bond, suchas a straight or branched group of 2-6 or 3-4 carbon atoms, referred toherein for example as C₂-C₆alkenyl, and C₃-C₄alkenyl, respectively.Exemplary alkenyl groups include, but are not limited to, vinyl, allyl,butenyl, pentenyl, etc.

The term “alkoxy” as used herein refers to a straight or branched alkylgroup attached to an oxygen (alkyl-O—). Exemplary alkoxy groups include,but are not limited to, alkoxys of 1-6 or 2-6 carbon atoms, referred toherein as C₁-C ₆alkoxy, and C₂-C₆ alkoxy, respectively. Exemplary alkoxygroups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

The term “alkenyloxy” used herein refers to a straight or branchedalkenyl group attached to an oxygen (alkenyl-O). Exemplary alkenoxygroupd include, but are not limited to, groups with an alkenyl group of3-6 carbon atoms, (also e.g. referred to as C₃-C₆alkenyloxy). Exemplary“alkenoxy” groups include, but are not limited to allyloxy, butenyloxy,etc.

The term “alkynyloxy” used herein refers to a straight or branchedalkynyl group attached to an oxygen (alkynyl-O)). Exemplary alkynyloxygroups include, but are not limited to, C₃-C₆ alkynyloxy, e.g.,propynyloxy.

The term “alkyl” as used herein refers to a saturated straight orbranched hydrocarbon, such as a straight or branched group of 1-6, 1-4,or 1-3 carbon atoms, referred to herein as C₁-C₆ alkyl, C₁-C₄ alkyl, andC₁-C₃ alkyl, respectively. Exemplary alkyl groups include, but are notlimited to, methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl,2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 3-methyl-2-butyl,2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl,4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl,2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,hexyl, etc. The term “haloalkyl” as used herein refers to a saturatedstraight or branched alkyl groups, in which one or more hydrogen atomsof the alkyl group are replaced with one or more independently selectedhalogens. The term “haloalkyl” encompasses alkyl groups in which all ofhydrogen atoms of the alkyl group are replaced independently selectedhalogens (sometimes referred to as “perhalo” alkyl groups. Exemplaryhaloalkyl groups include, but are not limited to, CH₂F, CH₂CH₂Cl, CF₃,CHFCH₂Cl.

The term “alkynyl” as used herein refers to an unsaturated straight orbranched hydrocarbon having at least one carbon-carbon triple bond, suchas a straight or branched group of 2-6, or 3-6 carbon atoms, referred toherein as C₂-C₆ alkynyl, and C₃-C₆alkynyl, respectively. Exemplaryalkynyl groups include, but are not limited to, ethynyl, propynyl,butynyl, pentynyl, hexynyl, methylpropynyl, etc.

The term “bridged cycloalkyl”, as used herein, is defined as amonocyclic 4- to 7-membered cycloalkyl group in which two non-adjacentatoms are linked by a CH₂ or CH₂CH₂ group. A “bridged cycloalkyl” may befused to one or more phenyl, partially unsaturated, or saturated rings.Examples of bridged carbocyclic groups include but are not limited tobicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, bicyclo[2.2.2]octene etc.

The term “carbonyl” as used herein refers to the radical —C(O)—. Theterm “cyano” as used herein refers to the radical —CN. The term “nitro”refers to the radical —NO₂. The term “H” refers to hydrogen.

The term “cycloalkoxy” as used herein refers to a cycloalkyl groupattached to an oxygen (cycloalkyl-O—).

The term “cycloalkyl” as used herein refers to a monocyclic saturated orpartically unsatured hydrocarbon group of for example 3-6, or 4-6carbons, referred to herein, e.g., as “C₃₋₆cycloalkyl” or “C₄₋₆cycloalkyl,” and derived from a cycloalkane. Exemplary cycloalkyl groupsinclude, but are not limited to, cyclohexyl, cyclohexenyl, cyclopentyl,cyclobutyl, cyclopropyl or cyclopentyl.

The terms “halo” or “halogen” as used herein refer to F, Cl, Br, or I.

The terms “heteroaryl” as used herein refers to a monocyclic aromatic4-6 membered ring system containing one or more heteroatoms, for exampleone to three heteroatoms, such as nitrogen, oxygen, and sulfur. Wherepossible, said heteroaryl ring may be linked to the adjacent radicalthough carbon or nitrogen. Examples of heteroaryl rings include but arenot limited to furyl, thienyl, pyrrolyl, thiazolyl, oxazolyl,isothiazolyl, isoxazolyl, imidazolyl, pyrazolyl, triazolyl, oxadiazolyl(e.g., 1,2,4-oxadiazolyl or 1,3,4-oxadiazolyl), pyridyl, andpyrimidinyl.

The terms “heterocyclyl” or “heterocyclic group” are art-recognized andrefer to saturated or partially unsaturated 4- to 7-membered ringstructures, whose ring structures include one to three heteroatoms, suchas nitrogen, oxygen, and sulfur. A heterocycle may be fused to one ormore phenyl, partially unsaturated, or saturated rings. Examples ofheterocyclyl groups include but are not limited to pyrrolidinyl,piperidinyl, morpholino, thiomorpholino, and piperazinyl.

The term “heterocyclylalkoxy” as used herein refers to aheterocyclyl-alkyl-O-group.

The term “heterocyclyloxyalkyl” refers to a heterocyclyl-O-alkyl-group.

The term “heterocycloxy” refers to a heterocyclyl-O-group. The term“cycloalkyloxy” refers to a cycloalkyl-O-group.

The term “heteroaryloxy” referes to a heteroaryl-O-group.

The terms “hydroxy” and “hydroxyl” as used herein refers to the radical—OH.

The term “oxo” as used herein refers to the radical ═O.

The term “nitrogen protecting group” or “amino protecting group” isart-recognized and as used herein refers to a chemical moiety that iscovalently linked to a nitrogen atom of an amino (primary or secondary)group and that temporarily blocks the reactivity of the amino groupduring a synthetic step and is selectively removed once the syntheticstep is complete. Nitrogen protecting groups include, for example,9-Fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc),carbobenzyloxycarbonyl (Cbz), p-methoxybenzyloxycarbonyl, acetyl,trifluoroacetyl, benzoyl, phthalimido, benzyl (Bn), p-methoxybenzyl,p-methoxyphenyl, 3,4-dimethoxybenzyl, triphenylmethyl, benzylidene, andp-toluenesulfonyl (Ts). In some embodiments, the nitrogen protectinggroup can have one of the following formulas: —C(O)OR₃₁ or —C(O)R₃₂ asdefined herein.

As used in the present disclosure, the term “partial NMDA receptoragonist” generally refers to a compound that is capable of binding to aglycine binding site of an NMDA receptor; at low concentrations a NMDAreceptor agonist acts substantially as agonist and at highconcentrations it acts substantially as an antagonist. Theseconcentrations are experimentally determined for each and every “partialagonist”.

“Pharmaceutically or pharmacologically acceptable” include molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, coatings, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions.

The term “pharmaceutical composition” as used herein refers to acomposition comprising at least one compound as disclosed hereinformulated together with one or more pharmaceutically acceptablecarriers.

“Individual,” “patient,” or “subject” are used interchangeably andinclude any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans. The compounds of the invention can beadministered to a mammal, such as a human, but can also be administeredto other mammals such as an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like). The mammal treated in themethods of the invention is desirably a mammal in which treatment e.g.,of pain or depression is desired. “Modulation” includes antagonism(e.g., inhibition), agonism, partial antagonism and/or partial agonism.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by the researcher, veterinarian, medical doctor or otherclinician. The compounds of the invention are administered intherapeutically effective amounts to treat a disease. Alternatively, atherapeutically effective amount of a compound is the quantity requiredto achieve a desired therapeutic and/or prophylactic effect, such as anamount which results in lessening a symptom of depression.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that are acidic in natureare capable of forming base salts with various pharmacologicallyacceptable cations. Examples of such salts include alkali metal oralkaline earth metal salts and, particularly, calcium, magnesium,sodium, lithium, zinc, potassium, and iron salts. Compounds included inthe present compositions that include a basic or acidic moiety may alsoform pharmaceutically acceptable salts with various amino acids. Thecompounds of the disclosure may contain both acidic and basic groups;for example, one amino and one carboxylic acid group. In such a case,the compound can exist as an acid addition salt, a zwitterion, or a basesalt.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as stereoisomers, such asgeometric isomers, enantiomers or diastereomers. The term“stereoisomers” when used herein consist of all geometric isomers,enantiomers or diastereomers. These compounds may be designated by thesymbols “R” or “S,” depending on the configuration of substituentsaround the stereogenic carbon atom. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.Stereoisomers include enantiomers and diastereomers. Mixtures ofenantiomers or diastereomers may be designated “(±)” in nomenclature,but the skilled artisan will recognize that a structure may denote achiral center implicitly.

The compounds of the disclosure may contain one or more chiral centersand/or double bonds and, therefore, exist as geometric isomers,enantiomers or diastereomers. The enantiomer and diastereomers may bedesignated by the symbols “(+),” “(−).” “R” or “S,” depending on theconfiguration of substituents around the stereogenic carbon atom, butthe skilled artisan will recognize that a structure may denote a chiralcenter implicitly. Geometric isomers, resulting from the arrangement ofsubstituents around a carbon-carbon double bond or arrangement ofsubstituents around a cycloalkyl or heterocyclic ring, can also exist inthe compounds of the present invention. The symbol

denotes a bond that may be a single, double or triple bond as describedherein. Substituents around a carbon-carbon double bond are designatedas being in the “Z” or “E” configuration wherein the terms “Z” and “E”are used in accordance with IUPAC standards. Unless otherwise specified,structures depicting double bonds encompass both the “E” and “Z”isomers. Substituents around a carbon-carbon double bond alternativelycan be referred to as “cis” or “trans,” where “cis” representssubstituents on the same side of the double bond and “trans” representssubstituents on opposite sides of the double bond. The arrangement ofsubstituents around a carbocyclic ring can also be designated as “cis”or “trans.” The term “cis” represents substituents on the same side ofthe plane of the ring and the term “trans” represents substituents onopposite sides of the plane of the ring. Mixtures of compounds whereinthe substituents are disposed on both the same and opposite sides ofplane of the ring are designated “cis/trans.”

The term “stereoisomers” when used herein consist of all geometricisomers, enantiomers or diastereomers. The present invention encompassesvarious stereoisomers of these compounds and mixtures thereof.

Individual enantiomers and diasteriomers of compounds of the presentinvention can be prepared synthetically from commercially availablestarting materials that contain asymmetric or stereogenic centers, or bypreparation of racemic mixtures followed by resolution methods wellknown to those of ordinary skill in the art. These methods of resolutionare exemplified by (1) attachment of a mixture of enantiomers to achiral auxiliary, separation of the resulting mixture of diastereomersby recrystallization or chromatography and liberation of the opticallypure product from the auxiliary, (2) salt formation employing anoptically active resolving agent, (3) direct separation of the mixtureof optical enantiomers on chiral liquid chromatographic columns or (4)kinetic resolution using steroselective chemical or enzymatic reagents.Racemic mixtures can also be resolved into their component enantiomersby well-known methods, such as chiral-phase gas chromatography orcrystallizing the compound in a chiral solvent. Stereoselectivesyntheses, a chemical or enzymatic reaction in which a single reactantforms an unequal mixture of stereoisomers during the creation of a newstereocenter or during the transformation of a pre-existing one, arewell known in the art. Stereoselective syntheses encompass both enantio-and diastereoselective transformations. For examples, see Carreira andKvaerno, Classics in Stereoselective Synthesis, Wiley-VCH: Weinheim,2009.

The compounds disclosed herein can exist in solvated as well asunsolvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms. In one embodiment, thecompound is amorphous. In one embodiment, the compound is a singlepolymorph. In another embodiment, the compound is a mixture ofpolymorphs. In another embodiment, the compound is in a crystallineform.

The invention also embraces isotopically labeled compounds of theinvention which are identical to those recited herein, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe invention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O,¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively. For example, a compoundof the invention may have one or more H atom replaced with deuterium.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Further, substitution with heavier isotopes such asdeuterium (i.e., ²H) may afford certain therapeutic advantages resultingfrom greater metabolic stability (e.g., increased in vivo half-life orreduced dosage requirements) and hence may be preferred in somecircumstances. Isotopically labeled compounds of the invention cangenerally be prepared by following procedures analogous to thosedisclosed in the e.g., Examples herein by substituting an isotopicallylabeled reagent for a non-isotopically labeled reagent.

The term “prodrug” refers to compounds that are transformed in vivo toyield a disclosed compound or a pharmaceutically acceptable salt,hydrate or solvate of the compound. The transformation may occur byvarious mechanisms (such as by esterase, amidase, phosphatase, oxidativeand or reductive metabolism) in various locations (such as in theintestinal lumen or upon transit of the intestine, blood or liver).Prodrugs are well known in the art (for example, see Rautio,Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). Forexample, if a compound of the invention or a pharmaceutically acceptablesalt, hydrate or solvate of the compound contains a carboxylic acidfunctional group, a prodrug can comprise an ester formed by thereplacement of the hydrogen atom of the acid group with a group such as(C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl havingfrom 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbonatoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N-(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Similarly, if a compound of the invention contains an alcohol functionalgroup, a prodrug can be formed by the replacement of the hydrogen atomof the alcohol group with a group such as (C₁-C₆)alkanoyloxymethyl,1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1((C₁-C₆)alkanoyloxy)ethyl(C₁-C₆)alkoxycarbonyloxymethyl, N-(C₁-C₆)alkoxycarbonylaminomethyl,succinoyl, (C₁-C₆)alkanoyl, α-amino(C₁-C₄)alkanoyl, arylacyl andα-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group isindependently selected from the naturally occurring L-amino acids,P(O)(OH)₂, —P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting fromthe removal of a hydroxyl group of the hemiacetal form of acarbohydrate).

If a compound of the invention incorporates an amine functional group, aprodrug can be formed, for example, by creation of an amide orcarbamate, an N-acyloxyakyl derivative, an (oxodioxolenyl)methylderivative, an N-Mannich base, imine or enamine. In addition, asecondary amine can be metabolically cleaved to generate a bioactiveprimary amine, or a tertiary amine can metabolically cleaved to generatea bioactive primary or secondary amine. For examples, see Simplicio, etal., Molecules 2008, 13, 519 and references therein.

Compounds

Disclosed compounds include those represented by the formula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl;    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, —OH,        C₁-C₆ alkoxy, —OC(O)—C₁-C₆ alkyl and —OC(O)-phenyl (optionally        substituted by one, two or three substituents selected from the        group consisting of halogen, hydroxyl, C₁-C₆ alkyl, and C₁-C₆        alkoxy);    -   R₄ is H or C₁-C₆ alkyl; and    -   X is selected from the group consisting of hydrogen,        —C₁₋₆alkylene-C₃₋₆cycloalkyl; C₁₋₆alkylene-heterocycle        (optionally substituted by one, two or three substituents        selected from the group consisting of halogen, hydroxyl, C₁-C₆        alkyl, and C₁-C₆ alkoxy), and —C₁₋₆alkylene-heteroaryl        (optionally substituted by one, two or three substituents        selected from the group consisting of halogen, hydroxyl,        C₁-C₆alkyl, and C₁-C₆alkoxy));        or in other embodiments, the variables set forth in formula (I)        are as defined as follows:    -   R_(b) is selected from the group consisting of H, halogen,        hydroxyl, cyano and C₁-C₆ alkyl (e.g., H);    -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆alkyl, —OH,        C₁-C₆alkoxy, —OC(O)—C₁-C₆ alkyl and OC(O)-phenyl (optionally        substituted by one, two or three substituents independently        selected from the group consisting of halogen, hydroxyl,        C₁-C₆alkyl, and C₁-C₆alkoxy);    -   R₄ is H or C₁-C₆ alkyl;    -   X is selected from the group consisting of:        -   (i) hydrogen;        -   (ii) —C₁₋₆alkylene-C₃₋₆cycloalkyl;        -   (iii) —C₁₋₆alkylene-heterocyclyl including from 3 to 6 ring            atoms wherein 1, 2, or 3 of the ring atoms are independently            selected from the group consisting of N, NH, N(C₁-C₃ alkyl),            O, and S; wherein the heterocyclyl is optionally substituted            by one, two or three substituents independently selected            from the group consisting of halogen, hydroxyl, C₁-C₆alkyl,            and C₁-C₆alkoxy);        -   (iv) —C₁₋₆alkylene-C(O)-heterocyclyl including from 3 to 6            ring atoms wherein 1, 2, or 3 of the ring atoms are            independently selected from the group consisting of N, NH,            N(C₁-C₃ alkyl), O, and S; wherein the heterocyclyl is            optionally substituted by one, two or three substituents            independently selected from the group consisting of halogen,            hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy);        -   (v) —C₁₋₆alkylene-heteroaryl including from 5 to 6 ring            atoms wherein 1, 2, or 3 of the ring atoms are independently            selected from the group consisting of N, NH, N(C₁-C₃ alkyl),            O, and S; wherein the heteroaryl is optionally substituted            by one, two or three substituents independently selected            from the group consisting of halogen, hydroxyl, C₁-C₆alkyl,            and C₁-C₆alkoxy;        -   (vi) branched unsubstituted C₃-C₆ alkyl; and        -   (vii) branched C₃-C₆ alkyl substituted with —C(O)NH₂ on one            carbon and —OH on another carbon;    -   and wherein the —NH₂ group attached to the carbon adjacent to        —CH(R₃)(R₄) is optionally substituted with a substituent        selected from —C(O)OR₃₁ and —C(O) R₃₂, wherein:    -   R₃₁ is selected from the group consisting of: C₁-C₆ alkyl; C₁-C₆        haloalkyl; C₂-C₆ alkenyl; C₂-C₆ alkynyl; C₃-C₁₀ cycloalkyl,        wherein the C₃-C₁₀ cycloalkyl is optionally substituted with        from 1-3 independently selected C₁-C₃ alkyl; —CH₂—C₃-C₁₀        cycloalkyl wherein the C₃-C₁₀ cycloalkyl is optionally        substituted with from 1-3 independently selected C₁-C₃ alkyl;        —CH₂-phenyl, wherein the phenyl is optionally substituted with        from 1-2 substituents independently selected from C₁-C₃ alkyl;        C₁-C₃ haloalkyl; C₁-C₃ alkoxy; C₁-C₃ haloalkoxy; nitro; halo;        SO₂Me, cyano; and —OC(O)CH₃; and —CH₂-pyridyl; and    -   R₃₂ is selected from the group consisting of: H; C₁-C₆ alkyl;        C₁-C₆ haloalkyl; phenyl, wherein the phenyl is optionally        substituted with from 1-2 substituents independently selected        from C₁-C₃ alkyl; C₁-C₃ haloalkyl; C₁-C₃ alkoxy; C₁-C₃        haloalkoxy; nitro; halo; SO₂Me, cyano; and —OC(O)CH₃; and        pyridyl.

In some embodiments, R₁ is H. In other embodiments, R₁ is C₁-C₆ alkyl(e.g., —CH₃).

In some embodiments, R₂ is H. In other embodiments , R₂ is C₁-C₆ alkyl(e.g., —CH₃).

In certain other embodiments, R₃ is —OH.

In some embodiments, R₄ is C₁-C₆ alkyl. In certain embodiments, R₄ ismethyl.

In some embodiments, X is as defined in definitions (i), (ii), (iii),and (v). In some embodiments, X is as defined in definitions (ii),(iii), (iv), and (v). In some embodiments, X is as defined indefinitions (ii), (iii), and (v). Embodiments in which X is as definedabove can include one or more of the following features: R₁ is H ormethyl (e.g., H); R₂ is H; R₃ is —OH; R₄ is methyl; R_(b) is H; the —NH₂group attached to the carbon adjacent to —CH(R₃)(R₄) is not substituted,the —NH₂ group attached to the carbon adjacent to —CH(R₃)(R₄) issubstituted with —C(O)OR₃₁, or the —NH₂ group attached to the carbonadjacent to —CH(R₃)(R₄) is substituted with —C(O)R₃₂.

In some embodiments, X is hydrogen.

In some embodiments, X is —C₁₋₆alkylene-heteroaryl (e.g.,C₁₋₂alkylene-heteroaryl or C₁ alkylene-heteroaryl), the heteroarylincluding from 5 to 6 ring atoms wherein 1, 2, or 3 of the ring atomsare independently selected from the group consisting of N, NH, N(C₁-C₃alkyl), O, and S; wherein the heteroaryl is optionally substituted byone, two or three substituents independently selected from the groupconsisting of halogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy. In certainembodiments, the heteroaryl is selected from the group consisting of1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, pyridyl, and pyrimidinyl. Incertain other embodiments, X is

Embodiments in which X is —C₁₋₆alkylene-heteroaryl can include one ormore of the following features: R₁ is H or methyl (e.g., H); R₂ is H; R₃is —OH; R₄ is methyl; R_(b) is H; the —NH₂ group attached to the carbonadjacent to —CH(R₃)(R₄) is not substituted, the —NH₂ group attached tothe carbon adjacent to —CH(R₃)(R₄) is substituted with —C(O)OR₃₁ (e.g.,R₃₁ is C₁-C₆ alkyl (e.g., tert-butyl)), or the —NH₂ group attached tothe carbon adjacent to —CH(R₃)(R₄) is substituted with —C(O)R₃₂ (e.g.,R₃₂ is C₁-C₆ alkyl (e.g., —CH₃ or iso-propyl)).

In some embodiments, X is hydrogen. In other embodiments, X isC₁₋₂alkylene-heteroaryl. In certain other embodiments, X is

In some embodiments, the —NH₂ group attached to the carbon adjacent to—CH(R₃)(R₄) is not substituted.

In other embodiments, the —NH₂ group attached to the carbon adjacent to—CH(R₃)(R₄) is substituted with a substituent selected from —C(O)OR₃₁and —C(O) R₃₂. In certain embodiments, the —NH₂ group attached to thecarbon adjacent to —CH(R₃)(R₄) is substituted with —C(O)OR₃₁. Inembodiments, R₃₁ is C₁-C₆ alkyl (e.g., tert-butyl). In otherembodiments, the —NH₂ group attached to the carbon adjacent to—CH(R₃)(R₄) is substituted with —C(O)R₃₂. In embodiments, R₃₂ is C₁-C₆alkyl (e.g., —CH₃ or iso-propyl).

In some embodiments, R₁ is methyl; R₂ is H; R₃ is —OH; R₄ is methyl; andX is hydrogen. In certain embodiments, the —NH₂ group attached to thecarbon adjacent to —CH(R₃)(R₄) is not substituted, the —NH₂ groupattached to the carbon adjacent to —CH(R₃)(R₄) is substituted with—C(O)OR₃₁ (e.g., R₃₁ is C₁-C₆ alkyl (e.g., tert-butyl)), or the —NH₂group attached to the carbon adjacent to —CH(R₃)(R₄) is substituted with—C(O)R₃₂ (e.g., R₃₂ is C₁-C₆ alkyl (e.g., —CH₃ or iso-propyl)). Incertain embodiments, R_(b) is H.

In some embodiments, R₁ is H; R₂ is H; R₃ is —OH; R₄ is methyl; and X ishydrogen. In certain embodiments, the —NH₂ group attached to the carbonadjacent to —CH(R₃)(R₄) is not substituted, the —NH₂ group attached tothe carbon adjacent to —CH(R₃)(R₄) is substituted with —C(O)OR₃₁ (e.g.,R₃₁ is C₁-C₆ alkyl (e.g., tert-butyl)), or the —NH₂ group attached tothe carbon adjacent to —CH(R₃)(R₄) is substituted with —C(O)R₃₂ (e.g.,R₃₂ is C₁-C₆ alkyl (e.g., —CH₃ or iso-propyl)). In certain embodiments,R_(b) is H.

In some embodiments, a disclosed compound includes any of thosedelineated in Table 1 and/or the Examples, e.g., one having the formula:

In another aspect, disclosed compounds include those represented by theformula:

and pharmaceutically acceptable salts, stereoisomers, and N-oxidesthereof, wherein

-   -   R₁ is H or C₁-C₆ alkyl;    -   R₂ is H or C₁-C₆ alkyl;    -   R₃ is selected from the group consisting of H, C₁-C₆ alkyl, —OH,        C₁-C₆ alkoxy, —OC(O)—C₁-C₆ alkyl and —OC(O)-phenyl;    -   R₄ is H or C₁-C₆ alkyl; and    -   X is —C₁-C₆alkylene-X′, wherein X′ is a 4- to 6-membered        heteroaryl having 1, 2, or 3 heteroatoms selected from O, N, or        S, where X′ is optionally substituted on a free carbon by one,        two or three substituents selected from the group consisting of        halogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy.

The compounds of the present disclosure and formulations thereof mayhave a plurality of chiral centers. Each chiral center may beindependently R, S, or any mixture of R and S. For example, in someembodiments, a chiral center may have an R:S ratio of between about100:0 and about 50:50, between about 100:0 and about 75:25, betweenabout 100:0 and about 85:15, between about 100:0 and about 90:10,between about 100:0 and about 95:5, between about 100:0 and about 98:2,between about 100:0 and about 99:1, between about 0:100 and 50:50,between about 0:100 and about 25:75, between about 0:100 and about15:85, between about 0:100 and about 10:90, between about 0:100 andabout 5:95, between about 0:100 and about 2:98, between about 0:100 andabout 1:99, between about 75:25 and 25:75, and about 50:50. Formulationsof the disclosed compounds comprising a greater ratio of one or moreisomers (i.e., R and/or S) may possess enhanced therapeuticcharacteristic relative to racemic formulations of a disclosed compoundsor mixture of compounds. In some instances, chemical formulas containthe descriptor “—(R)—” or “—(S)—” that is further attached to solidwedge or dashed wedge. This descriptor is intended to show a methanecarbon (CH) that is attached to three other substituents and has eitherthe indicated R or S absolute configuration (see, e.g., Table 1).

Disclosed compounds may provide for efficient cation channel opening atthe NMDA receptor, e.g. may bind or associate with the glutamate site ofthe NMDA receptor to assist in opening the cation channel. The disclosedcompounds may be used to regulate (turn on or turn off) the NMDAreceptor through action as an agonist.

The compounds as described herein may be glycine site NMDA receptorpartial agonists. A partial agonist as used in this context will beunderstood to mean that at a low concentration, the analog acts as anagonist and at a high concentration, the analog acts as an antagonist.Glycine binding is not inhibited by glutamate or by competitiveinhibitors of glutamate, and also does not bind at the same site asglutamate on the NMDA receptor. A second and separate binding site forglycine exists at the NMDA receptor. The ligand-gated ion channel of theNMDA receptor is, thus, under the control of at least these two distinctallosteric sites. Disclosed compounds may be capable of binding orassociating with the glycine binding site of the NMDA receptor. In someembodiments, disclosed compounds may possess a potency that is 10-foldor greater than the activity of existing NMDA receptor glycine sitepartial agonists.

The disclosed compounds may exhibit a high therapeutic index. Thetherapeutic index, as used herein, refers to the ratio of the dose thatproduces a toxicity in 50% of the population (i.e., TD₅₀) to the minimumeffective dose for 50% of the population (i.e., ED₅₀). Thus, thetherapeutic index=(TD₅₀):(ED₅₀). In some embodiments, a disclosedcompound may have a therapeutic index of at least about 10:1, at leastabout 50:1, at least about 100:1, at least about 200:1, at least about500:1, or at least about 1000:1.

Compositions

In other aspects, formulations and compositions comprising the disclosedcompounds and optionally a pharmaceutically acceptable excipient areprovided. In some embodiments, a contemplated formulation comprises aracemic mixture of one or more of the disclosed compounds.

Contemplated formulations may be prepared in any of a variety of formsfor use. By way of example, and not limitation, the compounds may beprepared in a formulation suitable for oral administration, subcutaneousinjection, or other methods for administering an active agent to ananimal known in the pharmaceutical arts.

Amounts of a disclosed compound as described herein in a formulation mayvary according to factors such as the disease state, age, sex, andweight of the individual. Dosage regimens may be adjusted to provide theoptimum therapeutic response. For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for themammalian subjects to be treated; each unit containing a predeterminedquantity of active compound calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier.

The specification for the dosage unit forms of the invention aredictated by and directly dependent on (a) the unique characteristics ofthe compound selected and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

The compounds can be administered in a time release formulation, forexample in a composition which includes a slow release polymer. Thecompounds can be prepared with carriers that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Many methods for the preparation of such formulations aregenerally known to those skilled in the art.

Sterile injectable solutions can be prepared by incorporating thecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In accordance with an alternative aspect of the invention, a compoundmay be formulated with one or more additional compounds that enhance thesolubility of the compound.

Methods

Methods for treating a condition in a patient in need thereof byadministering a therapeutically effective dose of a compound describedherein are provided. In some embodiments, the condition may be a mentalcondition. For example, a mental illness may be treated. In anotheraspect, a nervous system condition may be treated. For example, acondition that affects the central nervous system, the peripheralnervous system, and/or the eye may be treated. In some embodiments,neurodegenerative diseases may be treated.

In some embodiments, the methods include administering a compound totreat patients suffering from autism, anxiety, depression, bipolardisorder, attention deficit disorder, attention deficit hyperactivitydisorder (ADHD), schizophrenia, a psychotic disorder, a psychoticsymptom, social withdrawal, obsessive-compulsive disorder (OCD), phobia,post-traumatic stress syndrome, a behavior disorder, an impulse controldisorder, a substance abuse disorder (e.g., a withdrawal symptom, opiateaddiction, nicotine addiction, and ethanol addition), a sleep disorder,a memory disorder (e.g., a deficit, loss, or reduced ability to make newmemories), a learning disorder, urinary incontinence, multiple systematrophy, progressive supra-nuclear palsy, Friedrich's ataxia, Down'ssyndrome, fragile X syndrome, tuberous sclerosis,olivio-ponto-cerebellar atrophy, cerebral palsy, drug-induced opticneuritis, ischemic retinopathy, diabetic retinopathy, glaucoma,dementia, AIDS dementia, Alzheimer's disease, Huntington's chorea,spasticity, myoclonus, muscle spasm, Tourette's syndrome, epilepsy,cerebral ischemia, stroke, a brain tumor, traumatic brain injury,cardiac arrest, myelopathy, spinal cord injury, peripheral neuropathy,acute neuropathic pain, and chronic neuropathic pain.

In some embodiments, methods of treating a memory disorder associatedwith aging, schizophrenia, special learning disorders, seizures,post-stroke convulsions, brain ischemia, hypoglycemia, cardiac arrest,epilepsy, migraine, AIDS dementia, Huntington's chorea, Parkinson'sdisease, early stage Alzheimer's disease, and Alzheimer's disease arecontemplated.

In certain embodiments, methods for treating schizophrenia are provided.For example, paranoid type schizophrenia, disorganized typeschizophrenia (i.e., hebephrenic schizophrenia), catatonic typeschizophrenia, undifferentiated type schizophrenia, residual typeschizophrenia, post-schizophrenic depression, and simple schizophreniamay be treated using the methods and compositions contemplated herein.Psychotic disorders such as schizoaffective disorders, delusionaldisorders, brief psychotic disorders, shared psychotic disorders, andpsychotic disorders with delusions or hallucinations may also be treatedusing the compositions contemplated herein.

Paranoid schizophrenia may be characterized where delusions or auditoryhallucinations are present, but thought disorder, disorganized behavior,or affective flattening are not. Delusions may be persecutory and/orgrandiose, but in addition to these, other themes such as jealousy,religiosity, or somatization may also be present. Disorganized typeschizophrenia may be characterized where thought disorder and flataffect are present together. Catatonic type schizophrenia may becharacterized where the patient may be almost immobile or exhibitagitated, purposeless movement. Symptoms can include catatonic stuporand waxy flexibility. Undifferentiated type schizophrenia may becharacterized where psychotic symptoms are present but the criteria forparanoid, disorganized, or catatonic types have not been met. Residualtype schizophrenia may be characterized where positive symptoms arepresent at a low intensity only. Post-schizophrenic depression may becharacterized where a depressive episode arises in the aftermath of aschizophrenic illness where some low-level schizophrenic symptoms maystill be present. Simple schizophrenia may be characterized by insidiousand progressive development of prominent negative symptoms with nohistory of psychotic episodes.

In some embodiments, methods are provided for treating psychoticsymptoms that may be present in other mental disorders, including, butnot limited to, bipolar disorder, borderline personality disorder, drugintoxication, and drug-induced psychosis. In another embodiment, methodsfor treating delusions (e.g., “non-bizarre”) that may be present in, forexample, delusional disorder are provided.

Also provided are methods for treating social withdrawal in conditionsincluding, but not limited to, social anxiety disorder, avoidantpersonality disorder, and schizotypal personality disorder.

In some embodiments, methods are provided for treating neuropathic pain.The neuropathic pain may be acute or chronic. In some cases, theneuropathic pain may be associated with a condition such as herpes, HIV,traumatic nerve injury, stroke, post-ischemia, fibromyalgia, reflexsympathetic dystrophy, complex regional pain syndrome, spinal cordinjury, sciatica, phantom limb pain, diabetic neuropathy, and cancerchemotherapeutic-induced neuropathic pain. Methods for enhancing painrelief and for providing analgesia to a patient are also contemplated.

Further contemplated methods include a method of treating autism and/oran autism spectrum disorder in a patient need thereof, comprisingadministering an effective amount of a compound to the patient. In anembodiment, a method for reducing the symptoms of autism in a patient inneed thereof is contemplated, comprising administering an effectiveamount of a disclosed compound to the patient. For example, uponadministration, the compound may decrease the incidence of one or moresymptoms of autism such as eye contact avoidance, failure to socialize,attention deficit, poor mood, hyperactivity, abnormal sound sensitivity,inappropriate speech, disrupted sleep, and perseveration. Such decreasedincidence may be measured relative to the incidence in the untreatedindividual or an untreated individual(s).

Also provided herein is a method of modulating an autism target geneexpression in a cell comprising contacting a cell with an effectiveamount of a compound described herein. The autism gene expression may befor example, selected from ABAT, APOE, CHRNA4, GABRA5,GFAP, GRIN2A,PDYN, and PENK. In another embodiment, a method of modulating synapticplasticity in a patient suffering from a synaptic plasticity relateddisorder is provided, comprising administering to the patient aneffective amount of a compound.

In another embodiment, a method of treating Alzheimer's disease, ore.g., treatment of memory loss that e.g., accompanies early stageAlzheimer's disease, in a patient in need thereof is provided,comprising administering a compound. Also provided herein is a method ofmodulating an Alzheimer's amyloid protein (e.g., beta amyloid peptide,e.g. the isoform Aβ₁₋₄₂), in-vitro or in-vivo (e.g. in a cell)comprising contacting the protein with an effective amount of a compoundis disclosed. For example, in some embodiments, a compound may block theability of such amyloid protein to inhibit long-term potentiation inhippocampal slices as well as apoptotic neuronal cell death. In someembodiments, a disclosed compound may provide neuroprotective propertiesto a Alzheimer's patient in need thereof, for example, may provide atherapeutic effect on later stage Alzheimer's—associated neuronal celldeath.

In a further embodiment, a method of treating depression comprisingadministering a compound described herein is provided. In someembodiments, the treatment may relieve depression or a symptom ofdepression without affecting behavior or motor coordination and withoutinducing or promoting seizure activity. Exemplary depression conditionsthat are expected to be treated according to this aspect of theinvention include, but are not limited to, major depressive disorder,dysthymic disorder, psychotic depression, postpartum depression,premenstrual syndrome, premenstrual dysphoric disorder, seasonalaffective disorder (SAD), bipolar disorder (or manic depressivedisorder), mood disorder, and depressions caused by chronic medicalconditions such as cancer or chronic pain, chemotherapy, chronic stress,and post traumatic stress disorders. In addition, patients sufferingfrom any form of depression often experience anxiety. Various symptomsassociated with anxiety include fear, panic, heart palpitations,shortness of breath, fatigue, nausea, and headaches among others.Anxiety or any of the symptoms thereof may be treated by administering acompound as described herein.

Also provided herein are methods of treating a condition intreatment-resistant patients, e.g., patients suffering from a mental orcentral nervous system condition that does not, and/or has not,responded to adequate courses of at least one, or at least two, othercompounds or therapeutics. For example, provided herein is a method oftreating depression in a treatment resistant patient, comprising a)optionally identifying the patient as treatment resistant and b)administering an effective dose of a compound to said patient.

In some embodiments, a compound described herein may be used for acutecare of a patient. For example, a compound may be administered to apatient to treat a particular episode (e.g., a severe episode) of acondition contemplated herein.

Also contemplated herein are combination therapies comprising a compoundin combination with one or more other active agents. For example, acompound may be combined with one or more antidepressants, such astricyclic antidepressants, MAO-I's, SSRI's, and double and triple uptakeinhibitors and/or anxiolytic drugs. Exemplary drugs that may be used incombination with a compound include Anafranil, Adapin, Aventyl, Elavil,Norpramin, Pamelor, Pertofrane, Sinequan, Surmontil, Tofranil, Vivactil,Parnate, Nardil, Marplan, Celexa, Lexapro, Luvox, Paxil, Prozac, Zoloft,Wellbutrin, Effexor, Remeron, Cymbalta, Desyrel (trazodone), andLudiomill. In another example, a compound may be combined with anantipsychotic medication. Non-limiting examples of antipsychoticsinclude butyrophenones, phenothiazines, thioxanthenes, clozapine,olanzapine, risperidone, quetiapine, ziprasidone, amisulpride,asenapine, paliperidone, iloperidone, zotepine, sertindole, lurasidone,and aripiprazole. It should be understood that combinations of acompound and one or more of the above therapeutics may be used fortreatment of any suitable condition and are not limited to use asantidepressants or antipsychotics.

EXAMPLES

The following examples are provided for illustrative purposes only, andare not intended to limit the scope of the disclosure.

Table 1 below shows some exemplary compounds of the disclosure andprovides physiochemical characteristics of the compounds.

Molecular Compound Structure Weight cLogP tPSA Compound X

241 −1.74 95.66 Compound Y

323 −2.04 125.79 4S-FNL-1

227.2603 −2.15173 95.66 4S-FNL-2

327.3761 −0.659636 107.97 4S-FNL-16

241.2869 −1.73516 95.66 4S-FNL-14

241.2869 −1.73516 95.66 4S-FNL-20

323.3476 −2.7324 125.79 4S-FNL-18

352.4286 −1.98729 107.18 4S-FNL-15

352.4286 −1.98729 107.18 4S--FNL-3

442.3875 −3.31859 150.19 4S-FNL-19

409.4369 −0.960294 138.1 4S-FNL-10

351.3577 −3.33013 128.87 4S-FNL-7

409.4369 −1.65688 138.1 4S-FNL-9

379.4109 −2.08661 128.87 4S-FN-13

361.3956 −1.88186 115.73 4S-FNL-12

419.4748 −0.35934 124.96 4S-7

428.48 −1.82649 162.5 4S-40

309.3211 −3.14898 125.79 4S-17

319.3589 −1.75921 112.65 4S-FNL-4

369.4558 0.391768 99.18

Example 1 Synthesis of Compound X

Synthesis of tert-butyl(3-hydroxy-1-oxo-1-(1-oxo-2,5-diazaspiro [3.4]octan-5-yl)butan-2-yl)carbamate (2)

To a stirred solution of 1 (0.5 g, 3.96 mmol) in CH₂Cl₂ (20 mL) wereadded DIPEA (1.0 g, 7.92 mmol), A (0.869 mg, 3.96 mmol) and HATU (1.5 g,3.96 mmol) at RT under inert atmosphere. The resulting reaction mixturewas stirred for 2 h at RT; progress of the reaction was monitored byTLC. The reaction mixture was quenched with water and extracted withDCM. The organic layer was separated dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to obtain the crude.The crude product was purified via preparative HPLC to afford 2 (30 mg,2.3%) as syrup.

Synthesis of 5-(2-amino-3-hydroxybutanoyl)-2,5-diazaspiro [3.41]octan-1-one(Compound X)

To a stirred solution of 2 (30 mg, 0.09 mmol) in DCM (5 mL) was addeddioxane.HCl (2 mL) at RT and stirred for 2 h. The volatiles wereevaporated under reduced pressure. The crude was triturated with etherand pentane to afford Compound X (15 mg, 75%) as brown thick syrup.

Example 2 Synthesis of Compound Y

Synthesis of (2S,3R)-2-(((benzyloxy)carbonyl)amino)-3-hydroxybutanoicacid (4S-A)

To a stirring solution of NaHCO₃ (529 g, 6.30 mol) in water (1 L) wasadded L-threonine (250 g, 2.10 mol) at RT and stirred for 30 min. Thereaction mixture was cooled to 0° C. To this Cbz-Cl (850 mL, 2.52 mol,50% in PhCH₃) was added drop wise and stirred for 1 h. The reactionmixture was warmed to RT and again stirred for 28 h. To this MTBE (1L)was added and stirred for 20 min. Separated aqueous layer in toluene wasstirred for 20 min. Aqueous layer was acidified with 1N HCl (pH˜1-2) andextracted with EtOAc (3×1.5 L). The organic layer was washed with brine,dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Thecrude material was stirred with dicyclohexylamine (819 mL, 4.20 mol) for4 h to get white solid, filtered and dried. Obtained solid was refluxedwith EtOAc (1.5 L) for 1 h and then filtered. The solid material wasdissolved in water (1 L) and acidified with dilute H₂SO₄ and againstirred for 30 min. The aqueous layer was extracted with EtOAc (3×1 L).The separated organic layer was washed with brine, dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure. Obtained crudematerial was triturated with n-hexane to afford 4S-A (230 g, 43%) aswhite solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.55 (br m, 1H), 7.37-7.30 (m, 5H), 6.94(d, J=8.8 Hz, 1H), 5.05 (s, 2H), 4.08-3.94 (m, 2H), 1.02 (d, J=6.4 Hz,3H).

ELSD purity: 84.66%.

Synthesis of (2S,3R)-2-((tert-butoxycarbonyl)amino)-3-hydroxybutanoicacid (4S-B)

To a stirring solution of L-threonine (50 g, 420 mol) in THF/water (500mL/500 mL) were added NaHCO₃ (111 g, 1.05 mol) and stirred at RT for 30min. The reaction mixture was cooled to 0° C. and Boc-anhydride (137 mL,630 mmol) was added drop wise and the stirring was continued at RT for16 h. After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure and obtained residue wasdiluted with water (100 mL) and acidified by using 1N HCl (pH˜3). Theaqueous layer was extracted with EtOAc (2×250 mL). The combined organiclayer was washed with brine (1×200 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure to afford 4S-B (80 g,87%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ12.5 (br s, 1H), 6.30 (d, J=8.5 Hz, 1H),4.50 (br s, 1H), 4.05-4.02 (m, 1H), 3.88-3.86 (m, 1H), 1.39 (s, 9H),1.08 (d, J=6.0 Hz, 3H).

LCMS (m/z): 218.1 [M⁺-1].

Synthesis of(2S,3R)-3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)butanoic acid (4S-C)

To a stirring solution of compound 4S-B (40 g, 182 mmol) in DMF (400 mL)was added 60% NaH (18.2 g, 758 mmol) portion wise at −20° C. under N₂atmosphere and stirred for 2 h. To this was added benzyl bromide (66.8mL, 0.55 mol) dropwise and the reaction mixture was stirred at RT for 3h. After consumption of the starting material (by TLC), the reactionmixture was quenched with ice cold water and washed with diethyl ether(2×250 mL). The separated aqueous layer was acidified using citric acidsolution (100 mL) and extracted with EtOAc (2×250 mL). The combinedorganic layers were dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford compound 4S-C (45 g, 80%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ12.64 (br s, 1H), 7.34-7.25 (m, 5H), 6.46(d, J=8.5 Hz, 1H), 4.53 (d, J=11.5 Hz, 1H), 4.39 (d, J=12.0 Hz, 1H),4.00-3.98 (m, 2H), 1.39 (s, 9H), 1.15 (d, J=6.0 Hz, 3H).

Synthesis of (2S,3R)-benzyl3-(benzyloxy)-2-((tert-butoxycarbonyl)amino)butanoate(4S-D)

To a stirring solution of compound 4S-C (45 g, 146 mmol) in DMF (400 mL)was added K₂CO₃ (40 g, 292 mmol) under N₂ atmosphere and stirred for 30min. To this benzyl bromide (21 mL, 175 mmol) was added drop wise at 0°C. and stirred at RT for 16 h. The reaction mixture was quenched withice cold water and extracted with diethyl ether (2×250 mL). Theseparated organic layer was washed with brine, dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude material waspurified by silica gel column chromatography eluting with 20%EtOAc/n-hexane to afford compound 4S-D (48 g, 82%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.37-7.18 (m, 10H), 6.81 (d, J=9.0 Hz,1H), 5.08 (s, 2H), 4.49 (d, J=12.0 Hz, 1H), 4.32 (d, J=12.0 Hz, 1H),4.25-4.22 (m, 1H), 4.01-3.98 (m, 1H), 1.38 (s, 9H), 1.15 (d, J=6.0 Hz,3H).

Synthesis of (2S,3R)-benzyl2-amino-3-(benzyloxy)butanoate(4S-E)

To a stirring solution of compound 4S-D (48 g, 120 mmol) in diethylether (50 mL) was added diethyl ether saturated with HCl (350 mL) at 0°C. and stirred at RT for 10 h. After consumption of the startingmaterial (by TLC), the reaction mixture was concentrated under reducedpressure. The crude material was triturated with diethyl ether/n-pentane(50 mL/50 mL) and dried under reduced pressure to afford compound 4S-E(28 g, 77%) as semisolid (HCl salt).

¹H-NMR: (400 MHz, DMSO-d₆): δ8.59 (s, 2H), 7.50-7.25 (m, 10H), 5.23 (d,J=12.5 Hz, 1H), 5.16 (d, J=12.5 Hz, 1H), 4.54 (d, J=12.0 Hz, 1H), 4.36(d, J=12.0 Hz, 1H), 4.12-4.09 (m, 1H), 4.09-3.99 (m, 1H), 1.29 (d, J=6.5Hz, 3H).

Synthesis of(4S,5R)-Benzyl4-(chlorocarbonyl)-2,2,5-trimethyloxazolidine-3-carboxylate(4S-F)

To a stirring solution of (4S,5R)-3-((benzyloxy) carbonyl)-2,2,5-trimethyloxazolidine-4-carboxylic acid (0.6 g, 2.04 mmol) in DCM (10mL) was added oxalyl chloride (0.51 g, 4.08 mmol), at 0° C. under N₂atmosphere. The reaction mixture was stirred at RT for 30 min. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under N₂ atmosphere to afford 4S-F (crude, 0.6 g) as yellowsyrup was directly used for next step without purification.

Synthesis of(4S,5R)-3-(tert-butoxycarbonyl)-2,2,5-trimethyloxazolidine-4-carboxylicacid (4S-G)

To a stirring solution of 4S-B (20 g, 91.3 mmol) in THF (200 mL) wereadded 2, 2-dimethoxy propane (100 mL) followed by PPTS (2.29 g, 9.13mmol) at RT. The reaction mixture was heated to 80° C. for 16 h. Aftercompletion of starting material (by TLC), the reaction was evaporatedunder reduced pressure. The crude reaction mixture was diluted withwater (150 mL) and EtOAc (300 mL). The separated organic layer waswashed with (1×100 mL) of brine solution. The organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto afford 4S-G (19 g, 80.1. %) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 13.00 (br s, 1H), 4.07-4.04 (m, 1H),3.78-3.74 (m, 1H), 1.51 (s, 6H), 1.17 (d, J=7.0 Hz, 3H);

LCMS m/z: 260.3 [M⁺+1]

Synthesis of (2S,3R)-methyl2-amino-3-hydroxybutanoate (4S-H)

To a stirring solution of L-threonine (50 g, 420 mmol) in CH₃OH (250 mL)was added thionyl chloride (62.4 mL, 840 mmol) at 0° C. and stirred at75° C. for 6 h. After completion of starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford 4S-H(60 g, crude). This material was directly used for the next step withoutfurther purification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.45 (s, 2H), 5.70 (s, 1H), 4.12-4.10 (m,1H), 3.90 (s, 1H), 3.73 (s, 3H), 1.20 (d, J=6.5 Hz, 3H).

Synthesis of (2S,3R)-methyl2-(((benzyloxy)carbonyl)amino)-3-hydroxybutanoate (4S-I)

To a stirring solution of 4S-H (60 g, 353 mmol) in water/1,4 dioxane(150 mL/300 mL) The reaction mixture was cooled to 0° C. added NaHCO₃(88.9 g, 1.059 mol) at 0 C and stirred for 15 min. Cbz-Cl (60.7 mL, 426mmol) was added drop wise and stirred for 1 h. The reaction mixture wasstirred to RT and stirred for 12 h. After completion of startingmaterial (by TLC), diluted the reaction mass with EtOAc (300 ml). Theseparated organic layer was washed with (2×200 mL) of saturated NaHCO₃solution followed by brine solution (2×100 mL). The organic layer wasdried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford crude material was triturated with n-hexane (500mL)to afford 4S-I (70 g, 74%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.37-7.30 (m, 5H), 7.20 (d, J=8.4 Hz, 1H),5.06 (s, 2H), 4.78 (d, J=6.8 Hz, 1H), 4.09-4.05 (m, 2H), 3.64 (s, 3H),1.09 (d, J=6.0 Hz, 3H).

LCMS m/z: 268.2[M⁺1]

Synthesis of(2S,3R)-methyl-2-((benzyloxy)carbonyl)amino)-3-((tert-butyldimethylsilyl)oxy)butanoate(4S-J)

To a stirring solution of 4S-I (50 g, 187 mmol) in DMF (400 mL) wereadded DIPEA (86 mL, 468 mmol) TBDMS-Cl (33.66 mL, 224 mmol) at 0° C. andstirred at RT for 12 h. After completion of starting material (by TLC),diluted the reaction mass with EtOAc (500 ml). The separated organiclayer was washed with (2×200 mL) of water followed by brine solution(2×100 mL). The organic layer was dried over anhydrous Na₂SO₄, filteredand concentrated under reduced pressure to afford crude material, whichwas purified by column chromatography eluting 10% EtOAc/hexane to afford4S-J (50 g, 70.1%) as colorless syrup.

¹H-NMR: (400 MHz, CDCl₃): δ 7.39-7.32 (m, 5H), 5.43 (d, J=9.6 Hz, 1H),5.14 (s, 2H), 4.45-4.43 (m, 1H), 4.29-4.26 (m, 1H), 3.72 (s, 3H), 1.21(d, J=6.0 Hz, 3H), 0.83 (s, 9H), 0.09 (s, 6H) LCMS m/z: 382.2[M⁺+1]

Synthesis ofN-((benzyloxy)carbonyl)-O-(tert-butyldimethylsilyl)-L-threonine (4S-K)

To a stirring solution of 4S-J (2 g, 5.24 mmol) in THF/H₂O (20 mL/20 mL)were added LiOH.H₂O (659 mg, 15.74 mmol) at RT and stirred for 16 h.After consumption of the starting material (by TLC), the solvent fromthe reaction mixture was evaporated under reduced pressure. The cruderesidue was acidified using citric acid solution (pH˜4) and extractedwith EtOAc (2×250 mL). The combined organic layer was dried over Na₂SO₄and concentrated to afford 4S-K (1.7 g, 89.4%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.75 (br s, 1H), 7.37-7.31 (m, 5H), 6.74(d, J=9.2 Hz, 1H), 5.05 (s, 2H), 4.32-4.30 (m, 1H), 4.05-4.02 (m, 1H),1.11 (d, J=6.4 Hz, 3H), 0.82 (s, 9H), 0.02 (s, 6H);

LCMS (m/z): 368.5 [M⁺+1].

Synthesis of (tert-butoxycarbonyl)-L-serine (4S-L)

To a stirring solution of L-serine (30 g, 285 mmol) in 1, 4-dioxane (150mL)/water (150 mL) were added NaOH (24 g, 599 mmol). After addedBoc-anhydride (75 mL, 342 mmol) was added drop wise at 0° C. and stirredat RT for 16 h. After consumption of the starting material (by TLC), thereaction mixture was washed with EtOAc (2×150 mL). The aqueous layer pHwas acidified by using 1N HCl (pH˜4). The aqueous layer was extractedwith EtOAc (2×250 mL). The separated organic extracts were washed withbrine (1×150 mL) solution. The organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure to affordcompound 4S-L (38 g, 65%) as thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 6.67 (d, J=8.5 Hz, 1H), 3.98-3.94 (m, 1H),3.65-3.56 (m, 2H), 1.40 (s, 9H);

LCMS (ESI): m/z 206.1 [M⁻+1]

Synthesis of (S)-1-((benzyloxy)carbonyl)pyrrolidine-2-carboxylic acid(4S-M)

To a stirring solution of L-proline (250 g, 2.17 mol) in water (1 L) wasadded Na₂CO₃ (576 g, 5.43 mol) and stirred for 1 h. After being cooledto 0° C., benzylchloroformate(50% in PhCH₃) (444 g, 2.61 mol) was addeddrop wise to the reaction mixture and again stirred for 1 h. Theresulting reaction mixture was warmed to RT and further stirred for 24h. After consumption of the starting material (by TLC), the reaction wasdiluted with water (1 L) and ether (1.5 L). The separated aqueous layerwas treated with PhCH₃ (1.5 L) and acidified using 6NHCl. The aqueouslayer was extracted with EtOAc (3×1.5 L) combined organic extracts werewashed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to afford 4S-M (450 g, 84%) as lightyellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.71 (br s, 1H), 7.37-7.26 (m, 5H),5.07-4.99 (m, 2H), 4.25-4.15 (m, 1H), 3.45-3.34 (m, 2H), 2.25-2.14 (m,1H), 1.94-1.79 (m, 3H).

LCMS m/z: 250.4 [M⁺+1].

Synthesis of (S)-benzyl 2-(chlorocarbonyl)pyrrolidine-1-carboxylate(4S-N)

To a stirring solution of 4S-M (90 g, 361 mmol) in CH₂Cl₂ (400 mL) wasadded oxalyl chloride (42 mL, 542 mmol) at 0° C. and stirred for 2 h.After complete formation of acid chloride, the reaction mixture wasconcentrated under reduced pressure to afford 4S-N (95 g, crude). Thismaterial was directly used for the next step without furtherpurification.

Synthesis of 1,3,5-Tris(4-methoxybenzyl)-1,3,5-triazinane (4S-O)

To a stirring solution of (4-methoxyphenyl)methanamine (100 g, 72.9mmol) in EtOH (300 mL) at room temperature was added formaldehyde (33%aq, 56.2 mL) drop wise. The reaction mixture was stirred at roomtemperature for 1 h. After consumption of the starting material (byTLC), the reaction mixture was diluted with EtOAc (1000 mL) and washedwith water (1000 mL) followed by brine. The separated organic layer wasconcentrated under reduced pressure to obtain crude; which was finallywashed with n-hexane to afford compound 4S-O (98 g, 90.15%) as whitesolid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.18 (d, J=8.0 Hz, 6H), 6.81 (d, J=8.0 Hz,6H), 3.71 (s, 9H), 3.50 (s, 6H), 3.29 (s, 6H).

Synthesis of Benzyl 2-(4-methoxybenzyl)-1-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate (4S-P):

To a stirring solution of 4S-N (95.86 g (crude), 355 mmol) in dry CH₂Cl₂(400 mL) was cooled to −40° C. and added Et₃N (200 mL, 1.45 mol)dropwise. The reaction mixture was stirred at −40° C. for 45 min. Tothis a mixture of 4S-O (45 g, 100 mmol) in CH₂Cl₂ (150 mL) and BF₃OEt₂(37 mL, 302 mmol) was added drop wise at −40° C. The resulting reactionmixture was allowed to stirr at RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was washed withsaturated NaHCO₃ solution followed by brine. The separated organic layerwas dried over anhydrous Na₂SO₄ and concentrated under reduced pressure.The crude material was triturated with EtOAc (100 mL) to afford compound4S-P (71 g, 51.6%) as white crystalline solid.

¹H-NMR: (500 MHz, DMSO-d6): δ 7.36-7.30 (m, 5H), 7.24 (d, J=8.0 Hz, 1H),7.06 (d, J=8.0 Hz, 1H), 6.90 (d, J=7.5 Hz, 1H), 6.81 (d, J=8.5 Hz, 1H),5.09 (s, 2H), 4.29 (s, 1H), 4.13-3.96 (m, 1H), 3.73 (s, 3H), 3.11 (t,J=5.0 Hz, 2H), 2.16-2.09 (m, 2H), 1.83-1.77 (m, 2H), 1.20-1.15 (m, 2H).

LCMS m/z: 381 [M⁺+1].

Synthesis of Benzyl 1-oxo-2,5-diazaspiro [3.41] octane-5-carboxylate(4S-Q)

To a stirring solution of compound 4S-P (71 g, 186.8 mmol) in MeCN (710mL) and H₂O (355 mL) were cooled to 0° C. and added a solution of CAN(204.7 g, 373.6 mmol) in H₂O (71 mL). The reaction mixture was stirredat 0° C. for 1 h. The resulting mass was poured into ice cold water andthe aqueous layer was extracted with EtOAc (3×500 mL). The combinedorganic layers were washed with saturated NaHCO₃ solution (1×500 mL)followed by brine (1×500 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure to get crude. Obtained material waspurified by silica gel column chromatography eluting with 70%EtOAc/n-hexane to afford compound 4S-Q (31 g, 48.6%) as yellow thicksyrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.90 (s, 1H), 7.36-7.29 (m, 5H), 5.10 (s,2H), 3.53 (d, J=4.5 Hz, 2H), 3.36-3.30 (m, 1H), 3.17-3.13 (m, 1H),2.17-2.10 (m, 2H), 1.82-1.76 (m, 2H).

LCMS m/z: 261 [M⁺+1]

Synthesis of 2,5-diazaspiro [3.4] octan-1-one (4S-R)

To a stirring solution of compound 4S-Q (2 g, 8.01 mmol) in EtOAc (30mL) were added (50% wet) 10% Pd/C (600 mg) and stirred under H₂atmosphere (balloon pressure) for 3 h at RT. After completion ofreaction (by TLC), the reaction mixture was filtered through a pad ofcelite and washed with EtOAc (10 mL). The filtrate was concentratedunder reduced pressure to afford compound 4S-R (250 mg, 25%) ascolorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.67 (br s, 1H), 3.15-3.11 (m, 2H),2.90-2.81 (m, 2H), 1.90-1.87 (m, 2H), 1.74-1.63 (m, 3H).

Synthesis of ethyl pyrrolidine-2-carboxylate hydrochloride (4S-S)

To a stirring solution of L-proline (110 g, 956.5 mmol) in ethanol wasadded thionyl chloride (141 ml, 1911.3 mmol) and heated to 80° C. for 16h. The reaction mixture was brought to RT and concentrated under vacuumto afford compound 4S-S as hydrochloride salt (170 g, 99%).

¹H-NMR: (400 MHz, CDCl₃): δ 4.15-4.10 (m, 2H), 3.68-3.62 (m, 2H),3.59-3.47 (m, 2H), 2.49-2.37 (m, 1H), 2.27-2.05 (m, 3H), 1.18 (t, J=3.6Hz, 3H).

LCMS (m/z): 144 [M⁺+1].

Synthesis of 1-tert-butyl 2-ethyl pyrrolidine-1,2-dicarboxylate (4S-T)

To a stirring solution of compound 4S-S (70 g, 0.391 mol) in CH₂Cl₂ (700mL) were added Et₃N (170.7 mL, 1.22 mol) followed by Boc-anhydride (133g, 0.61 mol) at 0° C. The reaction mixture was stirred at RT for 12 h.After consumption of the starting material (by TLC), the reaction wasdiluted with water (100 mL) and extracted with CH₂Cl₂ (2×200 mL). Theorganic layer was washed with water (1×150 mL), brine (1×200 mL). Theorganic layer was dried over Na₂SO₄ and concentrated under reducedpressure to afford compound 4S-T (90 g, 90%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.15-4.10 (m, 2H), 4.09-4.02 (m, 1H),3.36-3.29 (m, 2H), 2.25-2.13 (m, 1H), 1.87-1.76 (m, 3H), 1.40 (s, 9H),1.18 (t, J=3.6 Hz, 3H).

LCMS (m/z): 144 [(M⁺1)-Boc].

HPLC: 96.11%.

Synthesis of 1-tert-butyl 2-ethyl2-((benzyloxy)methyl)pyrrolidine-1,2-dicarboxylate (4S-U)

To a stirring solution of compound 4S-T (100 g, 436 mmol) in THF (800mL) was added LiHMDS (873 mL, 960 mmol) at −40° C. and stirred for 1 h.To this BOM-chloride (95 mL, 655 mmol) was added drop wise at −50° C.and stirred for 1 h at RT. After consumption of the starting material(by TLC), the reaction was quenched with aqueous NH₄Cl solution andextracted with EtOAc (2×200 mL). The combined organic layer was washedwith citric acid solution (2×100 mL) followed by brine solution (2×100mL). The separated organic layer was dried over Na₂SO₄ and concentratedto afford compound 4S-U (150 g, 95%). This material was directly takenfor the next step without further purification.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.32-7.30 (m, 5H), 4.59 (s, 2H), 4.10-4.02(m, 2H), 4.09-4.00 (m, 2H), 3.30-3.24 (m, 2H), 2.21-2.10 (m, 1H),1.82-1.75 (m, 3H), 1.39 (s, 9H), 1.18 (t, J =3.6 Hz, 3H).

LCMS (m/z): 263.4 [(M⁺+1)-Boc].

Synthesis of2-((benzyloxy)methyl)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylicacid (4S-V)

To a stirring solution of compound 4S-U (150 g, 429 mmol) in methanol(200 mL), THF (100 ml) was added NaOH solution (24 g in 300 mL H₂O) atRT. The reaction mixture was heated to 65° C. for 16 h. Afterconsumption of the starting material (by TLC), the solvent from thereaction was evaporated under reduced pressure and diluted with EtOAc(2×200 mL). The aqueous layer was acidified using citric acid solution(pH˜3) and extracted with EtOAc (2×250 mL). The combined organic layerwas dried over Na₂SO₄ and concentrated to afford compound 4S-V (75 g,52%).

¹H-NMR: (400 MHz, CDCl₃): δ 7.37-7.32 (m, 5H), 4.61 (s, 2H), 4.05-3.88(m, 2H), 3.65-3.42 (m, 2H), 2.54-2.46 (m, 2H), 1.95 (br s, 2H), 1.57 (s,9H).

LCMS (m/z): 335.3 [M⁺+1].

Synthesis of1-(tert-butoxycarbonyl)-2-(hydroxymethyl)pyrrolidine-2-carboxylic acid(4S-W)

To a stirring solution of compound 4S-V (75 g, 223 mmol) in methanol(750 mL) was added 50% wet 10% Pd/C (20 g) at RT and stirred for 6 hunder H₂ atmosphere. After consumption of the starting material (byTLC), the reaction mixture was filtered through a pad of celite and thepad was washed with methanol (50 mL). Obtained filtrate was concentratedunder reduced pressure to afford compound 4S-W (50 g, 91.1%) as paleyellow solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 12.55 (br s, 1H), 3.99 (d, 1H), 3.88 (d,1H), 7.65-7.60 (m, 1H), 3.51-3.45 (m, 1H), 3.39-3.34 (m, 1H), 2.32-2.14(m, 1H), 1.98-1.69 (m, 3H), 1.39 (s, 9H).

LCMS (m/z): 246.12 [M⁺+1].

Synthesis of benzyl 6-methyl-1-oxo-2,5-diazaspiro [3.41]octane-5-carboxylate (4S-AE) Synthesis of ethyl5-oxopyrrolidine-2-carboxylate (4S-X)

To a stirring solution of 5-oxopyrrolidine-2-carboxylic acid (10 g, 77.4mmol) in ethanol (100 mL) was added thionyl chloride (6.7 mL, 92.9 mmol)at 0° C. The reaction mixture was stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the solvents from thereaction mixture were removed under vacuum. The residue was diluted withEtOAc (50 mL) and stirred over K₂CO₃. The organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography to affordcompound 4S-X (9 g, 74%).

¹H-NMR: (400 MHz, DMSO-d₆): δ7.98 (br s, 1H), 4.16 (t, 3H), 2.37-2.30(m, 1H), 2.15 (q, 2H), 2.03-1.97 (m, 1H), 1.22 (t, 3H).

LCMS m/z: 157.9 [M⁺+1].

Synthesis of 1-tert-butyl 2-ethyl 5-oxopyrrolidine-1,2-dicarboxylate(4S-Y)

To a stirring solution of compound 4S-X (9 g, 57.3 mmol) in CH₂Cl₂ (90mL) was added DMAP (7.0 g, 57.3 mmol) followed by Et₃N (15.9 mL, 114.6mmol) and Boc-anhydride (36.7 mL, 171.9 mmol) at 0° C. The reactionmixture was stirred at RT for 16 h. The reaction mixture was dilutedwith CH₂Cl₂ (50 mL) and washed with aqueous citric acid solutionfollowed by brine. The separated organic layer was dried over anhydrousNa₂SO₄ and concentrated under vacuum. Obtained crude material waspurified by column chromatography eluting with 50% EtOAc/Hexane toafford compound 4S-Y (12 g, 82%).

¹H-NMR: (400 MHz, DMSO-d₆): δ4.61 (m, 1H), 4.19 (q, 2H), 2.46-2.40 (m,2H), 2.37-2.25 (m, 1H), 1.91-1.85 (m, 1H), 1.42 (s, 9H), 1.22 (t, 3H).

Synthesis of ethyl 2-((tert-butoxycarbonyl)amino)-5-oxohexanoate (4S-Z)

To a stirring solution of compound 4S-Y (12 g, 46.6 mmol) in THF (120mL) under inert atmosphere was added MeMgBr (3M in ether) (20.2 mL, 60.6mmol) at 0° C. and stirred for 2 h. After consumption of the startingmaterial (by TLC), the reaction mixture was quenched with aqueous NH₄Clsolution and the aqueous layer was extracted with EtOAc (2×200mL). Thecombined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude residue was purified bysilica gel column chromatography eluting with 20% EtOAc/Hexane to affordcompound 4S-Z (10 g, 79%).

¹H-NMR: (400 MHz, CDCl₃): δ5.14 (br s, 1H), 4.23 (q, 2H), 2.62-2.47 (m,2H), 2.17 (s, 4H), 1.91-1.82 (m, 1H), 1.45 (s, 10H), 1.26 (t, 3H).

Synthesis of ethyl 5-methylpyrrolidine-2-carboxylate (4S-AA & 4S-AB)

To a stirring solution of compound 4S-Z (10 g, 36.7 mmol) in CH₂Cl₂ (100mL) was added TFA (14.89 mL, 194.6 mmol) at 0° C. After being stirredfor 2 h at RT, the reaction mixture was concentrated under reducedpressure to get compound 4S-AA (crude). This was dissolved in ethanol(100 mL) and added 10% Pd/C (50% wet, 3 g) under N2 atmosphere. Thereaction mixture was stirred under H₂ atmosphere (balloon pressure) for16 h. The reaction mixture was filtered through a pad of celite andfiltrate was concentrated under reduced pressure to afford compound4S-AB (15 g, crude). This material was directly taken for the next stepwithout further purification.

¹H-NMR: (500 MHz, DMSO-d₆): 4.4 (m, 1H), 4.2 (m, 2H), 3.6 (m,1H), 2.3(m, 1H), 2.1 (m, 2H), 1.6 (m, 1H), 1.3 (d, 3H), 1.2 (t, 3H).

LCMS m/z: 158.1 [M⁺+1].

Synthesis of 1-benzyl 2-ethyl 5-methylpyrrolidine-1,2-dicarboxylate(4S-AC)

To a stirring solution of compound 4S-AB (15 g, 95.4 mmol) in1,4-dioxane/water (150 mL, 1: 1) was added NaHCO₃ (24 g, 286.3 mmol)followed by Cbz-Cl (50% in PhCH₃, 143.1mmol) at 0° C. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (100 mL)and extracted with EtOAc (2×100mL). The separated organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatography20% EtOAc/hexane as eluent to afford compound 4S-AC (9 g, 32%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.41-7.25 (m, 5H), 4.52 (s, 2H), 4.35-4.32(m, 1H), 4.16-4.10 (m, 3H), 2.24-2.20 (m, 1H), 2.11-2.05 (m, 1H),1.97-1.93 (m, 1H), 1.64-1.60 (m, 1H), 1.26-1.22 (m, 4H), 1.12 (t, 2H).

LCMS m/z: 292 [M⁺+1].

Synthesis of benzyl 2-(4-methoxybenzyl)-6-methyl-1-oxo-2,5-diazaspiro[3.4] octane-5-carboxylate (4S-AD)

To a stirring solution of compound 4S-AC (10 g, 34.1 mmol) in dry THF(50 mL) was added LiHMDS (1M in THF, 102.2 mL, 0.102 mol) at −10° C.under N₂ atmosphere and stirred for 1 h. To this a solution of 4S-AQ(6.55 g, 37.4 mmol) in THF (50 mL) was added and stirred for 2 h. Afterconsumption of the starting material (by TLC), the reaction was quenchedwith aqueous NH₄Cl solution and extracted with EtOAc (2×100 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure. Obtained crude material was purified by silicagel column chromatography eluting with 30% EtOAc/hexane to affordcompound 4S-AD (6 g, 45%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.41-7.37 (m, 4H), 7.22 (d, 2H), 7.15-7.07(m, 1H), 6.92-6.80 (m, 2H), 5.19-4.90 (m, 1H), 4.15-4.12 (m, 1H),4.01-3.98 (m, 1H), 3.75 (d, 3H), 3.49-3.45 (m, 1H), 3.16-3.12 (m, 1H),2.31-2.27 (m, 1H), 2.05-1.99 (m, 1H), 1.67-1.60 (m, 1H), 1.17 (d, 3H).

Synthesis of benzyl 6-methyl-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-AE)

To a stirring solution of compound 4S-AD (4 g, 10.14mmol) in H₂O/ACN(120 mL, 1:1) was added CAN (16.6 g, 30.4 mmol) at 0° C. The reactionmixture was stirred at RT for 3 h. After consumption of the startingmaterial (by TLC), the reaction was diluted with water (50 mL) andextracted with EtOAc (2×50 mL). The separated organic layer was washedwith aqueous NaHCO₃ solution, dried over anhydrous Na₂SO₄ andconcentrated under vacuum. Obtained crude material was purified bycolumn chromatography using 30% EtOAc/hexane as eluent to affordcompound 4S-AE (1 g, 36%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.95 (d, 1H), 7.37-3.35 (m, 5H), 5.18-5.04(m, 2H), 4.07-3.95 (m, 1H), 3.64-3.53 (m, 1H), 3.15-3.07 (m, 1H),2.33-1.99 (m, 3H), 1.59-1.50 (m, 1H), 1.15 (t, 3H).

LCMS m/z: 273.2 [M⁺−1].

Synthesis of (S)-1-benzyl 2-ethyl pyrrolidine-1,2-dicarboxylate (4S-AF)

To a stirred solution of compound 4S-S (140 g, 782 mmol) in 1,4dioxane/water (700 mL/700 mL) were added NaHCO₃ (197 g, 2.34 mol),Cbz-Cl (235 g, 1.17 mol) at 0° C. After the reaction mixture was stirredat RT for 16 h. After completion of starting material (by TLC), thereaction mixture was diluted with EtOAc (300 mL). The separated organiclayer was dried over anhydrous Na₂SO₄ and concentrated under reducedpressure to obtained crude compound was purified by columnchromatography to afford compound 4S-AF (130 g, 60%) as pale yellowliquid.

¹H-NMR: (500 MHz, CDCl₃): δ 7.37-7.26(m, 5H), 5.05 (s, 2H), 4.39-4.31(m, 1H), 4.22-4.18 (m, 1H), 4.07-4.04 (m, 1H), 3.62 (t, J=4.5 Hz, 2H),2.02-1.87(m, 4H), 1.27 (t, J=7.5 Hz, 3H).

Mass m/z: 278 [M⁺+1].

Synthesis of 1-Benzyl 2-ethyl 2-acetylpyrrolidine-1,2-dicarboxylate(4S-AG)

To a stirring solution of 4S-AF (20 g, 72.1 mmol) in THF (100mL) wasadded LiHMDS (86.5 mL, 86.54 mmol, 1M solution in THF) at −20° C. andstirred for 1 h under inert atmosphere. To this acetyl chloride (5.66mL, 79.33 mmol) was slowly added and stirring was continued for another1 h at −20° C. The reaction mixture was quenched with aqueous NH₄Clsolution and then extracted with EtOAc. Combined organic extracts weredried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Thecrude material was purified by silica gel column chromatography elutingwith 10% EtOAc/Hexane to afford compound 4S-AG (14 g, 60.8%) ascolorless thick syrup.

Mass m/z: 320 [M⁺+1].

Synthesis of 1-Benzyl 2-ethyl2-(1-aminoethyl)pyrrolidine-1,2-dicarboxylate (4S-AH)

To a stirring solution of compound 4S-AG (14 g, 43.88 mmol) in EtOH (140mL) was added ammonium acetate(16.9 g, 219.4 mmol) followed by sodiumcyanoborohydride (8.2 g, 131.6 mmol) at RT under inert atmosphere. Theresultant reaction mixture was heated to 60° C. and stirred for 16 h.The progress of the reaction was monitored by TLC and the volatiles wereevaporated under reduced pressure. The residue was diluted with EtOAcand washed with water. The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The crude was purified bysilica gel column chromatography eluting with 2% MeOH/CH₂Cl₂ to affordcompound 4S-AH (0.6 g, 55%) as colorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.86 (br s, 2H), 7.37-7.28 (m, 5H), 5.16(s, 2H), 4.10-4.01 (m, 1H), 3.95-3.90 (m, 2H), 3.73-3.69 (m, 1H),3.47-3.42 (m, 1H), 2.10-1.90 (m, 4H), 1.31 (d, J=12.0 Hz, 3H), 1.05 (t,J=7.5 Hz, 3H).

LCMS (ESI): 321.3 [M⁺+1].

Synthesis of benzyl 1-methyl-3-oxo-2,5-diazaspiro [3.41]octane-5-carboxylate (4S-AI)

To a stirring solution of compound 4S-AH (8.0 g, 25 mmol) in THF (150mL) was added t-butylmagnesium chloride (1M in THF) (75 mL, 75 mmol) at0° C. under nitrogen atmosphere slowly over a period of 15 min andstirred for 2 h at 0° C. After completion of reaction by TLC, thereaction mixture was quenched with aqueous NH₄Cl solution and theaqueous layer was extracted with EtOAc (2×50 mL). The separated organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to obtain crude product, which was purified by silicagel column chromatography eluting with 2% MeOH/DCM to afford compound4S-AI (4.5 g, 65.6%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.08-8.03 (m, 1H), 7.39-7.30 (m, 5H),5.13-5.00 (m, 2H), 4.05-4.02 (m, 1H), 3.53-3.47 (m, 1H), 3.41-3.36 (m,1H), 2.10-2.05 (m, 1H), 1.96 (s, 1H), 1.90-1.85 (m, 2H), 1.22 (t, J=6.8Hz, 3H).

LCMS (ESI): 275.2 [M⁺+1].

Synthesis of methyl pyrrolidine-2-carboxylate (4S-AJ)

To a stirring solution of L-proline (100 g, 0.87 mol) in methanol (800mL) was added thionyl chloride (76.9 mL, 1.04 mol) slowly dropwise at 0°C. The reaction mixture was heated to 80° C. for 12 h. After consumptionof the starting material (by TLC), the reaction was concentrated undervacuum. Obtained residue was washed with n-Hexane (200 mL) and distilledoff the solvent to afford compound 4S-AJ (143.9 g, HCl salt).

¹H-NMR: (400 MHz, CDCl₃): δ 3.89 (s, 3H), 3.68-3.62 (m, 2H), 3.59-3.47(m, 2H), 2.49-2.37 (m, 1H), 2.27-2.05 (m, 3H).

LCMS (ESI): 130 [M⁺+1].

Synthesis of 1-tert-butyl 2-methyl pyrrolidine-1,2-dicarboxylate (4S-AK)

To a stirring solution of compound 4S-AJ (35 g, 0.22 mol) in CH₂Cl₂ (175mL) were added Et₃N (90 mL, 0.65 mol) followed by Boc-anhydride (56.9mL, 0.26 mol) at 0° C. The reaction mixture was stirred at RT for 16 h.After consumption of the starting material (by TLC), the reaction wasdiluted with water (100 mL) and extracted with CH₂Cl₂ (2×100 mL). Theorganic layer was washed with water (1×100 mL), brine (1×100 mL) and theseparated organic layer was dried over Na₂SO₄ and concentrated. Obtainedcrude material was purified by silica gel column chromatography elutingwith 30% EtOAc/Hexane to afford compound 4S-AK (41 g, 95%).

¹H-NMR: (400 MHz, CDCl₃): δ 4.25-4.21 (m, 1H), 3.75 (s, 3H), 3.57-3.26(m, 2H), 2.29-2.10 (m, 1H), 1.99-1.75 (m, 3H), 1.45 (s, 9H).

LCMS (ESI): 130 [(M⁺+1)-Boc]

Synthesis of 1-tert-butyl 2-methyl 2-acetylpyrrolidine-1,2-dicarboxylate(4S-AL)

To a stirring solution of compound 4S-AK (40g, 0.17mol) in THF (200 mL)was added LiHMDS (183.4 mL, 0.18 mol, 1M solution in THF) at −20° C. andstirred for 30 min under inert atmosphere. To this acetyl chloride(12.46mL, 0.17mol) was slowly added and stirring was continued foranother 1 h at −20° C. After completion of the reaction, quenched withaqueous NH₄Cl solution (100 mL) and then extracted with EtOAc (2×200mL). Combined organic extracts were dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford compound 4S-AL (35 g,crude)(mixture of mono and di acetylated compound) as yellow thicksyrup.

¹H-NMR: (500 MHz, CDCl₃): δ 3.79 (s, 3H), 3.64-3.38 (m, 2H), 2.43-2.17(m, 3H), 2.03 (s, 3H), 1.48-1.42 (m, 1H), 1.39 (s, 9H).

Mass m/z: 272 [M⁺+1].

Synthesis of 1-tert-butyl 2-methyl2-(1-aminoethyl)pyrrolidine-1,2-dicarboxylate (4S-AM)

To a stirring solution of compound 4S-AL (35 g, crude, 0.12 mol) in EtOH(350 mL) was added ammonium acetate(49.7 g, 0.64 mol) at RT and heatedto 70° C. for 1 h. After reaction mass temperature was cooled to RT andadded sodium cyanoborohydride (16.2 g, 0.25 mol) and the resultantreaction mixture was heated to 75° C. for 16 h. The progress of thereaction was monitored by TLC, and the volatiles were evaporated underreduced pressure. The residue was diluted with EtOAc (200 mL) and washedwith water (2×100 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The crude was purified bysilica gel column chromatography eluting with 2% MeOH/CH₂Cl₂ to affordcompound 4S-AM (17 g, 48.4%) as colorless syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.69 (br s, 2H), 4.05-3.99 (m, 2H), 3.87(s, 3H), 3.71-3.60 (m, 1H), 2.24-2.11 (m, 1H), 1.99-1.71 (m, 3H), 1.42(s, 9H), 1.07 (d,J=6.8 Hz, 3H).

LCMS (ESI): 273 [M⁺+1].

Synthesis of tert-butyl 1-methyl-3-oxo-2,5-diazaspiro[3.4]octane-5-carboxylate (4S-AN)

To a stirring solution of compound 4S-AM (1.0 g, 3.67 mmol) in THF (15mL) was added t-butylmagnesium chloride (1M in THF) (11 mL, 11.02 mmol)at 0° C. under nitrogen atmosphere slowly over a period of 15 min andstirred for 2 h at 0° C. After completion of reaction by TLC, thereaction mixture was quenched with aqueous NH₄Cl solution (20 mL) andthe aqueous layer was extracted with EtOAc (2×30 mL). The combinedorganic layer was dried over anhydrous Na₂SO₄, filtered and concentratedunder reduced pressure to obtain compound 4S-AN (800 mg, crude) whichwas used directly for next step without purification.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.97 (d, J=9.2 Hz, 1H), 3.93-3.91 (m, 1H),3.72-3.55 (m, 1H), 3.28-3.24 (m, 1H), 2.08-1.98 (m, 1H), 1.86-1.75 (m,2H), 1.67-1.64 (m, 1H), 1.39 (s, 9H), 1.18 (d, J=4.4 Hz, 3H).

LCMS (ESI): 241 [M⁺+1].

Synthesis of tert-butyl2-(2-ethoxy-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-AO)

To a stirring solution of compound 4S-AN (6.7 g, 27.8 mmol) in CH₃CN (70mL) were added ethyl 2-bromoacetate(4.98 mL, 41.8 mmol), cesiumcarbonate(27.2 g, 83.6 mmol) and stirred at RT for 16 h. Afterconsumption of the starting material (by TLC), the reaction was dilutedwith water (50 mL) and extracted with EtOAc (2×75 mL). The combinedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to afford compound 4S-AO (8.6 g, crude), which was useddirectly for next step without any purification.

LCMS m/z: 327.3 [M⁺+1].

Synthesis of pyrimidin-2-ylmethanamine (4S-AP)

To a stirring solution of pyrimidine-2-carbonitrile (2.0 g, 19.0 mmol)in methanol (50 mL) were added 10% Pd/C (300 mg), 12 N HCl (1.5 mL)under N₂ atmosphere. The reaction mixture was stirred under H₂atmosphere (balloon pressure) at RT for 3 h. After consumption of thestarting material (by TLC), the reaction mixture was filtered through apad of celite and the pad was washed with methanol. Obtained filtratewas concentrated under reduced pressure to afford crude compound whichwas triturated with diethyl ether to obtained compound 4S-AP (1.2 g,44%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.87 (d, J=5.0 Hz, 2H), 8.69 (br s, 2H),7.52 (t, J=5.0 Hz, 1H), 4.24 (s, 2H).

Mass (ESI) (m/z): 110.3 [M⁺+1].

Synthesis of 2-((4-methoxybenzyl)amino)acetonitrile (4S-AQ)

To a stirring solution of (4-methoxyphenyl)methanamine (35 g, 255 mmol)in CH₂Cl₂ (350 mL) were added Et₃N (52.3 mL, 383 mmol) andbromoacetonitrile (21.2 mL, 306 mmol) at 0° C. under nitrogenatmosphere. The reaction mixture was stirred at RT for 16 h. Thereaction was diluted with CH₂Cl₂ (150 mL) and washed with brine. Theseparated organic layers were dried over Na₂SO₄ and concentrated underreduced pressure. The crude residue was purified by columnchromatography using 30% EtOAc/Hexane as eluent to afford compound 4S-AQ(22 g, 49%).

¹H-NMR: (500 MHz, CDCl₃): δ 7.27 (d, 2H), 6.90 (d, 2H), 3.87 (s, 2H),3.84 (s, 3H), 3.56 (s, 2H).

Synthesis of 2-chloro-1-(pyrrolidin-1-yl) ethanone (4S-AR)

To a stirring solution of pyrrolidine (5 g, 70.42 mmol) in DCM (30 mL)was added to chloroacetyl chloride (7.8 g, 70.42 mmol), at 0° C. underN₂ atmosphere. The reaction mixture was stirred at RT for 12 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (25 mL). The organic layer was separated, washed withwater (100 mL), and dried over anhydrous Na₂SO₄ concentrated underreduced pressure to afford 4S-AR as a reddish solid (6 g, 60%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 4.26 (s, 2H), 3.43 (t, J=7.0 Hz, 2H), 3.29(t, J=7.5 Hz, 2H), 1.89-1.84 (m, 2H), 1.79-1.73 (m, 2H).

LCMS m/z: 148.3 [M⁺+1].

Synthesis of Benzyl 2-(2-ethoxy-2-oxoethyl)-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate 4S-AS

To a stirred solution of compound 4S-Q (31 g, 119 mmol) in acetonitrile(310 mL) were added Cs₂CO₃ (97 g, 297.8 mmol) and ethyl2-bromoacetate(19.6 mL, 178.7 mmol) at RT and stirred for 16 h at RT.After completion of the reaction, volatiles were evaporated underreduced pressure. The residue was diluted with water (250 mL) andextracted with EtOAc (2×300 mL). The separated organic layer was washedwith brine, dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The obtained crude material was purified by silica gel columnchromatography eluting with 50% EtOAc/Hexane to afford compound 4S-AS(29 g, 70.2%) as yellow thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.35-7.30 (m, 5H), 5.06 (s, 2H), 4.21 (s,1H), 4.18 (s, 1H), 4.13-4.10 (m, 2H), 3.69 (d, J=4.5 Hz, 1H), 3.47-3.44(m, 3H), 2.16 (t, J=6.0 Hz, 2H), 1.87-1.80 (m, 2H), 1.21-1.14 (m, 3H).

LCMS m/z: 347 [M⁺+1].

Synthesis of Benzyl 2-(2-hydrazinyl-2-oxoethyl)-1-oxo-2,5-diazaspiro[3.4] octane-5-carboxylate (4S-AT)

To a stirred solution of compound 4S-AS (29 g, 83.7 mmol) in EtOH (580mL) was added hydrazine hydrate(12.2 mL, 251 mmol) at RT and stirred at90° C. for 2 h. After completion of starting material (by TLC), ethanolwas evaporated under reduced pressure. The crude residue was purified bycolumn chromatography by eluting with 2% MeOH/DCM to afford compound4S-AT (19 g, 68%) as colorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.10 (s, 1H), 7.38-7.32 (m, 5H), 5.10-4.99(m, 2H), 4.30-4.15 (m, 2H), 3.81 (s, 2H), 3.66-3.42 (m, 2H), 3.40-3.30(m, 2H), 2.22-2.08 (m, 2H), 1.87-1.84 (m, 2H).

LCMS m/z: 333.3 [M⁺+1].

Synthesis of Benzyl2-((1,3,4-oxadiazol-2-yl)methyl)-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-AU)

To a stirred solution of compound 4S-AT (19 g, 57.1 mmol) in triethylorthoformate (190 mL) was added p-TSA (1.08 g, 5.71 mmol) at RT andstirred at 80° C. for 2 h. After completion of starting material (byTLC), triethyl orthoformate was evaporated under reduced pressure. Thecrude residue was purified by column chromatography eluting 1% MeOH/DCMto afford compound 4S-AU (11 g, 56.2%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): 9.17 (s, 1H), 7.37-7.30 (m, 5H), 5.06(s,2H), 4.67-4.59 (m, 1H), 4.39-3.67 (m, 1H), 3.57-3.48 (m, 1H),3.46-3.44 (m, 1H), 3.40-3.31 (m, 2H), 2.21-1.98 (m, 2H), 1.87-1.80 (m,2H).

LCMS m/z: 343.4[M⁺+1].

Synthesis of 2-((1,3,4-oxadiazol-2-yl)methyl)-2,5-diazaspiro [3.4]octan-1-one 4S-AV

To a stirring solution of compound 4S-AU (1 g, 2.92 mmol) in methanol(20 mL) was added 10% wet Pd/C (300 mg) and stirred under H₂ atmosphere(balloon pressure) for 12 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (10 mL). Thefiltrate was concentrated under reduced pressure to afford 4S-AV (280mg, 46%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ9.22 (s, 1H), 4.67 (s, 2H), 3.36 (s, 2H),3.29-3.23 (m, 1H), 3.07-2.84 (m, 2H), 1.93-1.90 (m, 2H), 1.76-1.65 (m,2H).

LCMS m/z: 208.2 [M⁺+1].

HPLC: 97.32% (both isomers).

Synthesis of benzyl ((2S,3R)-3-hydroxy-1-oxo-1-(1-oxo-2,5-diazaspiro[3.4] octan-5-yl)butan-2-yl) carbamate (4S-1)

To a stirring solution of compound 4S-R (250 mg, 1.98 mmol) in DCM (20mL) were added N, N-diisopropylethylamine (0.91 mL, 4.95 mmol), 4S-A(602 mg, 2.38 mmol), followed by HOBt (321 mg, 2.38 mmol), EDCI (366 mg,2.38 mmol), at 0° C. and stirred at RT for 16 h. After consumption ofthe starting material (by TLC), the reaction mixture was diluted withwater (10 mL). The separated organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure to obtained crudecompound which was purified by column chromatography to afford compound4S-1 (200 mg, 28%) as colorless syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ7.85 (s, 1H), 7.36-7.31 (m, 5H), 5.04 (s,2H), 4.72 (d, J=6.4 Hz, 2H), 4.11-4.09 (m, 1H), 4.03-3.99 (m, 1H),3.82-3.75 (m, 1H), 3.70-3.63 (m, 1H), 3.02-2.75 (m, 2H), 1.97 (t, J=2.8Hz, 1H), 1.90-1.78 (m, 3H), 1.18-1.13 (m, 3H); LCMS m/z: 444.5 [M⁺+Na].

Synthesis of 5-((2S,3R)-2-amino-3-hydroxybutanoyl)-2,5-diazaspiro [3.4]octan-1-one (4S-FNL-1)

To a stirring solution of compound 4S-1 (400 mg, 1.10 mmol) in EtOAc (20mL) was added (50% wet) 10% Pd/C (200 mg) and stirred under H₂atmosphere (balloon pressure) for 16 h at RT. After completion ofreaction (by TLC), the reaction mixture was filtered through a pad ofcelite and triturated with EtOAc (10 mL). The filtrate was concentratedunder reduced pressure to afford compound (4S-FNL-1) (90 mg, 35.8%) ascolorless thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.37 (d, J=2.4 Hz, 1H), 7.59 (s, 1H), 4.72(d, J=6.4 Hz, 2H), 4.11-4.09 (m, 1H), 4.03-3.99 (m, 1H), 3.82-3.75 (m,1H), 3.70-3.63 (m, 1H), 3.02-2.75 (m, 2H), 1.97 (t, J=2.8 Hz, 1H),1.90-1.78 (m, 3H), 1.18-1.13 (m, 3H).

LCMS m/z: 228.2 [M⁺+1].

HPLC: 90.8%.

Synthesis of tert-butyl (3-hydroxy-1-oxo-1-(1-oxo-2,5-diazaspiro [3.4]octan-5-yl)butan-2-yl)carbamate (4S-FNL-2)

To a stirred solution of 4S-R (0.5 g, 3.96 mmol) in CH₂Cl₂ (20 mL) wereadded DIPEA (1.0 g, 7.92 mmol), 4S-B (0.869 mg, 3.96 mmol) and HATU (1.5g, 3.96 mmol) at RT under inert atmosphere. The resulting reactionmixture was stirred for 2 h at RT; progress of the reaction wasmonitored by TLC. The reaction mixture was quenched with water andextracted with DCM. The organic layer was separated dried over anhydrousNa₂SO₄, filtered and concentrated under reduced pressure to obtain thecrude. The crude product was purified via preparative HPLC to afford(4S-FNL-2) (30 mg, 2.3%) as syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 8.23 (br s, 1H), 8.03 (br s, 1H), 6.89 (brs, 1H), 3.98-3.96 (m, 2H), 3.76-3.74 (m, 2H), 3.62-3.57 (m, 1H),2.92-2.87 (m, 1H), 1.78-1.74 (m, 2H), 1.68-1.62 (m, 2H), 1.38 (s, 9H),1.14-0.92 (m, 3H).

Synthesis of tert-butyl 2-(((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)carbamoyl)-2-(hydroxymethyl) pyrrolidine-1-carboxylate (4S-2)

To a stirring solution of compound 4S-W (10 g, 40.8 mmol) in CH₂Cl₂ (100mL) were added DIPEA (22 mL, 122 mmol), 4S-E (12 g, 40.8 mmol), HATU (23g, 61.2 mmol) at RT and stirred for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(50 mL) and extracted with CH₂Cl₂ (2×100 mL). The combined organic layerwas washed with brine, dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting with 30%EtOAc/n-hexane to afford compound 4S-2 (15g, 79.3%) as pale yellowliquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.25-8.12 (m, 1H), 7.31-7.27 (m, 10H),5.85 (t, J=4.8 Hz, 1H), 5.14 (s, 2H), 4.54-4.49 (m, 2H), 4.31-4.20 (m,1H), 4.15-4.07 (m, 1H), 3.91-3.50 (m, 1H), 3.52-3.37 (m, 1H), 3.31-3.27(m, 2H), 2.35-2.07 (m, 1H), 1.95-1.90 (m, 1H), 1.73-1.52 (m, 2H), 1.39(s, 9H), 1.19 (d, J=6.4 Hz, 3H).

Mass (ESI): m/z 527.4 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-1,3-bis(benzyloxy)-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro[3.4] octane-5-carboxylate (4S-3)

To a stirring solution of triphenylphosphine (1.5 g, 5.69 mmol) in THF(10 mL) was added DIAD (0.91 g, 4.55 mmol) at RT and stirred for 30 min.To this added compound 4S-2 (1.2 g, 2.27 mmol) in (10 mL) THF slowly andreaction mixture was stirred at RT for 2 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography eluting 30% EtOAc/n-hexane to afford compound 4S-3 (1.0g, 90%) as pale yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.33-7.26 (m, 5H), 7.23-7.18 (m, 5H), 5.10(s, 2H), 4.80-4.73 (m,2H), 4.60 (s, 2H), 4.31 (s, 2H), 4.05-4.00 (m,2H), 1.80-1.68 (m, 4H), 1.39 (s, 9H), 1.18 (d, J=6.0 Hz, 3H).

Mass (ESI): m/z 509.4 [M⁺+1].

Synthesis of (2S,3R)-2-(5-(tert-butoxycarbonyl)-1-oxo-2,5-diazaspiro[3.4] octan-2-yl)-3-hydroxybutanoic acid (4S-4)

To a stirring solution of compound 4S-3 (10 g, 19.64 mmol) in methanol(100 mL) was added 10% Pd/C (4 g) at RT and stirred for 24h under H₂atmosphere. After consumption of the starting material (by TLC), thereaction mixture was filtered through a pad of celite and the pad waswashed with methanol (50mL). Obtained filtrate was concentrated underreduced pressure to obtained crude, which was triturated with n-pentane(50 mL) to afford compound 4S-4 (6 g, 93.7%) as white solid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 12.80 (br s, 1H), 4.78-4.73 (m, 1H),4.21-4.19 (m, 1H), 4.09 (s, 2H), 3.55-3.46 (m, 2H), 2.09-2.05 (m, 2H),1.80 (d, J=7.0 Hz, 1H), 1.38 (s, 9H), 1.35-1.28 (m, 2H), 1.17 (d, J=6.5Hz, 3H).

LCMS (M/Z) m/z: 329.6 [M⁺+1].

Synthesis of tert-butyl2-((2S,3R)-1-amino-3-hydroxy-1-oxobutan-2-yl)-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-5)

To a stirring solution of compound 4S-4 (500 mg, 1.52 mmol) in CH₂Cl₂(10 mL) were added DIPEA (0.8 mL, 4.57 mmol), EDCI.HCl (350 mg, 1.82mmol) followed by HOBt (280 mg, 1.82 mmol), NH₄Cl (161 mg, 3.04 mmol) at0° C. and stirred for 16 h at RT. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (10 mL)and extracted with CH₂Cl₂ (2×30 mL). The combined organic layer waswashed with citric acid solution (2×30 mL). The organic layer was driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressure.Obtained crude material was purified by silica gel column chromatographyeluting 2% MeOH/DCM to afford compound 4S-5 (200 mg, 40%) as colorlessliquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.53 (s, 2H), 4.59 (s, 1H), 4.02 (s, 1H),3.77-3.70 (m, 2H), 3.62-3.53 (m, 2H), 3.46-3.33 (m, 1H), 2.17-2.03 (m,2H), 1.88-1.71 (m, 2H), 1.38 (s, 9H), 1.18 (d, J=6.5 Hz, 3H).

Mass (ESI): m/z 328.3 [M⁺+1].

Synthesis of (2S,3R)-3-hydroxy-2-(1-oxo-2,5-diazaspiro [3.4] octan-2-yl)butanamide (4S-6)

To a stirring solution of compound 4S-5 (200 mg, 0.61 mmol) in CH₂Cl₂ (5mL) was added TFA (0.5 mL, 6.1 mmol) at 0° C. and stirred at RT for 3 h.After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to obtained crude compound which wastriturated with n-pentane/diethyl ether (5 mL/5 mL) to afford compound4S-6 (100 mg, crude) as white solid (TFA salt).

¹H-NMR: (400 MHz,D₂O): δ 4.37-4.29 (m, 2H), 4.13-4.07 (m, 1H), 3.98-3.95(m, 1H), 3.59-3.50 (m, 2H), 2.51-2.44 (m, 2H), 2.26-2.19 (m, 2H), 1.32(d, J=6.0 Hz, 3H).

Mass (ESI): m/z 228.2 [M⁺+1].

Synthesis of(2S,3R)-3-hydroxy-2-(5-((2S,3R)-3-hydroxy-2-methylbutanoyl)-1-oxo-2,5-diazaspiro[3.4] octan-2-yl) butanamide (4S-7)

To a stirring solution of compound 4S-6 (1.2 g, 3.70 mmol) in CH₂Cl₂ (20mL) were added DIPEA (1.92 mL, 11.1 mmol), 4S-B (810 mg, 3.70 mmol),HATU (2.1 g, 5.55 mmol) at 0° C. and stirred for 16 h at RT. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with water (20 mL) and extracted with CH₂Cl₂ (2×40 mL). Thecombined organic layer was dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. Obtained crude material waspurified by silica gel column chromatography eluting 5% MeOH/DCM toafford compound 4S-7 (250 mg, crude) as semi solid.

¹H-NMR: (400 MHz, CD₃OD): δ 4.58-4.40 (m, 1H), 4.29-4.22 (m, 2H),4.03-3.93 (m, 3H), 3.80-3.66 (m, 2H), 3.51-3.46 (m, 1H), 2.28-2.25 (m,2H), 2.07-2.01 (m, 2H), 1.44 (s, 9H), 1.38 (d, J=2.8 Hz, 2H), 1.20 (d,J=6.0 Hz, 3H), 1.17 (d, J=6.8 Hz, 3H).

Mass (ESI): m/z 328.3 [M⁺+1].

Synthesis of(2S,3R)-2-(5-((2S,3R)-2-amino-3-hydroxybutanoyl)-1-oxo-2,5-diazaspiro[3.4] octan-2-yl)-3-hydroxybutanamide (4S-FNL-3)

To a stirring solution of compound 4S-7 (200 mg (crude), 0.46 mmol) inCH₂Cl₂ (10 mL) was added TFA (0.4 mL, 4.67 mmol) at 0° C. and stirredfor 2 h at RT. After completion of reaction (by TLC), the reactionmixture was concentrated under reduced pressure to obtained crudecompound which was triturated with DCM/diethyl ether (5 mL/5 mL) toafford (4S-FNL-3) (150 mg, 99.3%) as white solid (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 4.40-4.37 (m, 1H), 4.31-4.27 (m, 3H), 4.08 (d,J=6.0 Hz, 1H), 3.88-3.68 (m, 3H), 2.43-2.34 (m, 2H), 2.31-2.26 (m, 2H),1.33 (d, J=6.4 Hz, 3H), 1.28 (d, J=6.8 Hz, 3H).

LCMS (ESI): m/z 328.3 [M⁺+1].

Synthesis of tert-butyl2-(hydroxymethyl)-2-(isobutylcarbamoyl)pyrrolidine-1-carboxylate (4S-8)

To a stirring solution of compound 4S-W (2.0 g, 8.16 mmol) in CH₂Cl₂ (30mL) was added DIPEA (4.25 mL, 24.4 mmol), 2-methylpropan-1-amine (0.97mL, 9.79 mmol) followed by HATU (4.65 g, 12.24 mmol) at 0° C. Thereaction mixture was warmed to RT and stirred for 16 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with H₂O (20 mL). The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget crude product, which was purified by silica gel columnchromatography eluting with 50% EtOAc/hexane to afford compound 4S-8(1.8 g, 73.46%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.65-7.56 (m, 1H), 5.03-4.94 (m, 1H),4.04-4.00 (m, 2H), 3.85-3.81 (m, 1H), 3.66-3.50 (m, 3H), 2.28-2.23 (m,1H), 1.87-1.69 (m, 4H), 1.38 (s, 9H), 0.83 (d, J=7.0 Hz, 6H)

LCMS (ESI): m/z 301.4 [M⁺+1]

Synthesis of tert-butyl 2-isobutyl-1-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-9)

To a stirring solution of TPP (3.92 g, 14.98 mmol)) in THF (20 mL) wasadded DTAD (2.37 mL, 11.98 mmol) at RT and stirred for 20 min. Compound4S-8 was added (1.8 g, 5.99 mmol) and the reaction stirred at RT for 4h. After consumption of the starting material (by TLC), the reactionmixture was concentrated under reduced pressure to get crude product,which was purified by silica gel column chromatography eluting with 40%EtOAc/hexane to afford compound 4S-9 (1.4 g, 82.8%) as thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.91-4.73 (m, 2H), 3.59-3.43 (m, 1H),3.37-3.26 (m, 2H), 3.17-3.11 (m, 1H), 2.14-2.07 (m, 2H), 1.84-1.71 (m,3H), 1.39 (s, 9H), 0.90-0.82 (m, 6H)

LCMS (m/z): 283.3 [M⁺+1]

Synthesis of 2-isobutyl-2,5-diazaspiro [3.4] octan-1-one (4S-10)

To a stirring solution of compound 4S-9 (1.0 g, 3.54 mmol) in DCM (10mL) was added TFA (2.72 mL, 35.41 mmol) at 0° C. under N₂ atmosphere andstirred at RT for 2 h. After consumption of the starting material (byTLC), the reaction mixture was concentrated under reduced pressure toobtain crude product, which was triturated with n-pentane (10 mL) toafford compound 4S-10 (1 g, crude) was used directly for next stepwithout any purification.

¹H-NMR: (400 MHz, DMSO-d₆): δ 4.90-4.75 (m, 1H), 3.67-3.50 (m, 2H),3.37-3.23 (m, 2H), 3.04-2.94 (m, 2H), 2.28-2.13 (m, 2H), 2.03-1.96 (m,2H), 1.87-1.80 (m, 1H), 0.86 (d, J=6.4 Hz, 6H);

LCMS (ESI): m/z 183.3 [M⁺+1]

Synthesis of tert-butyl((2S)-3-hydroxy-1-(2-isobutyl-1-oxo-2,5-diazaspiro [3.4]octan-5-yl)-1-oxopropan-2-yl)carbamate (4S-FNL-4)

To a stirring solution of compound 4S-10 (1.0 g, 3.37 mmol) in CH₂Cl₂(20 mL) was added DIPEA (2.9 mL, 16.87 mmol), 4S-L (899 mg, 4.38 mmol)followed by HATU (1.92 g, 5.06 mmol) at RT and stirred for 16 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with H₂O (20 mL). The separated organic layer was dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toget crude product, which was purified by silica gel columnchromatography eluting with 2% MeOH/DCM to afford (4S-FNL-4) (350 mg,28.2%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 6.88-6.69 (m, 1H), 4.88-4.77 (m, 1H),4.39-4.33 (m, 1H), 3.69-3.49 (m, 5H), 3.43-3.37 (m, 1H), 2.92-2.89 (m,2H), 2.09-2.04 (m, 2H), 1.90-1.76 (m, 3H), 1.38 (s, 9H), 0.86 (d, J=6.8Hz, 6H);

HPLC: 93.12%

LCMS (ESI): m/z 370.4 [M⁺+1]

Synthesis oftert-butyl((2S,3R)-1-(2-((1,3,4-oxadiazol-2-yl)methyl)-1-oxo-2,5-diazaspiro[3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl)carbamate (4S-FNL-7)

To a stirring solution of compound 4S-AV (1 g, 4.8 mmol) in DMF (20 mL)were added DIPEA (4.4 mL, 24.03 mmol), 4S-B (1.26 g, 5.76 mmol), HATU(2.73 g, 7.2 mmol) at RT and stirred for 16 h at RT. After consumptionof the starting material (by TLC), the reaction mixture was diluted withwater (30 mL) and extracted with EtOAc (2×30 mL). The combined organiclayer was washed with water (2×50 mL) followed by brine solution (2×50mL). The separated organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with2% MeOH/DCM followed by preparative HPLC purification to afford(4S-FNL-7) (280 mg, 14.2%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.24 (s, 1H), 6.67 (d, J=6.4 Hz, 1H), 4.80(s, 2H), 4.76-4.70 (m, 1H), 4.24-4.21 (m, 1H), 4.06-4.01 (m, 2H),3.84-3.79 (m, 2H), 3.73-3.56 (m, 1H), 2.18-1.98 (m, 2H), 1.91-1.86 (m,2H), 1.38 (s, 9H), 1.03 (d, J=6.4 Hz, 3H).

Mass (ESI): m/z 410.4 [M⁺+1].

HPLC: 99.73% (both isomers).

Synthesis of benzyl((2S,3R)-1-(2-((1,3,4-oxadiazol-2-yl)methyl)-1-oxo-2,5-diazaspiro [3.4]octan-5-yl)-3-hydroxy-1-oxobutan-2-yl)carbamate (4S-13)

To a stirring solution of 4S-AV (1.2 g, 5.76 mmol) in DMF (15 mL) wereadded DIPEA (3 mL, 17.3 mmol), 4S-A (1.75 g, 6.91 mmol), HATU (3.28 g,8.64 mmol) at 0° C. and stirred to RT for 16 h. After consumption of thestarting material (by TLC), the reaction mixture was diluted with water(100 mL) and EtOAc (100 mL). The organic layer was washed with brinesolution (2×100 mL). The organic layer was dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. Obtained crudematerial was purified by silica gel column chromatography eluting with2% MeOH/DCM to afford compound 4S-13 (800 mg, 31.3%) as an off-whitesolid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.24 (s, 1H), 7.36-7.31 (m, 5H), 7.11 (d,J=8.5 Hz, 1H), 5.07-4.99 (m, 2H), 4.81-4.74 (m, 2H), 4.60-4.57 (m, 1H),4.29-4.10 (m, 1H), 3.85-3.71 (m, 2H), 3.63-3.55 (m, 2H), 2.16-2.09 (m,2H), 2.07-1.86 (m, 2H), 1.19-1.12 (m, 3H).

LCMS (ESI): m/z 444.4 [M⁺+1].

Synthesis of2-((1,3,4-oxadiazol-2-yl)methyl)-5-(L-threonyl)-2,5-diazaspiro [3.4]octan-1-one (4S-40):

To a stirring solution of compound 4S-13 (800 mg, 1.8 mmol) in methanol(20 mL) was added 10% wet Pd/C (240 mg) and stirred under H₂ atmosphere(balloon pressure) for 12 h at RT. The reaction mixture was filteredthrough a pad of celite and triturated with methanol (10 mL). Thefiltrate was concentrated under reduced pressure to afford crudecompound which was purified by column chromatography by eluting 8%MeOH/DCM to afford compound 4S-40 (400 mg, 72%) as white colorsemisolid.

¹H-NMR: (400 MHz, DMSO-d₆): 9.12 (s, 1H), 7.54 (s, 2H), 5.19 (d, J=4.4Hz, 1H), 4.09-3.97 (m, 1H), 3.90-3.72 (m, 1H), 3.66-3.60 (m, 2H),3.26-3.13 (m, 2H), 2.88-2.74 (m, 2H), 2.12-1.92 (m, 1H), 1.89-1.74 (m,3H), 1.16-1.08 (m, 3H).

LCMS m/z: 208.2 [M⁺+1].

HPLC: 97.63% (both isomers).

Synthesis ofN-((2S,3R)-1-(2-((1,3,4-oxadiazol-2-yl)methyl)-1-oxo-2,5-diazaspiro[3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl)isobutyramide (4S-FNL-9)

To a stirred solution of 4S-40 (400 mg, 1.29 mmol) in DCM (5 mL) wasadded triethyl amine (0.36mL, 2.58 mmol) at 0° C. After added isobutyrylchloride (0.15 mL, 1.41 mmol) at 0° C. and stirred at RT for 2 h. Aftercompletion of starting material (by TLC), reaction mass was evaporatedunder reduced pressure. The crude residue was purified by columnchromatography eluting 4% MeOH/DCM followed by preparative HPLCpurification to afford (4S-FNL-9) (55 mg, 11.2%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): 9.20 (s, 1H), 8.28 (s, 1H), 5.26-5.09 (m,1H), 4.99-4.48 (m, 2H), 4.10-3.91 (m, 1H), 3.89-3.71 (m, 3H), 3.69-3.61(m, 1H), 3.41-3.31 (m, 1H), 2.82-2.49 (m, 1H), 2.24-2.18 (m, 1H),1.98-1.73 (m, 3H), 1.10 (d, J=6.4 Hz, 3H), 0.97-0.93 (m, 6H).

LCMS m/z: 380.4[M⁺+1].

HPLC: 97.84%.

Synthesis ofN-((2S,3R)-1-(2-((1,3,4-oxadiazol-2-yl)methyl)-1-oxo-2,5-diazaspiro[3.41] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl) acetamide (4S-FNL-10)

To a stirring solution of compound (4S-40) (800 mg, 2.58 mmol) in DCM(15 mL) was added TEA (783 mg, 7.74 mmol) followed by acetyl chloride(0.36 mL, 5.16 mmol) at 0° C. and stirred for 1 h. After consumption ofthe starting material (by TLC), the reaction mass was quenched withwater (2 mL). The reaction mixture was concentrated under reducedpressure to obtain crude product which was purified by silica gel columnchromatography eluting with 5% MeOH/DCM, preparative HPLC followed bychiral preparative HPLC purification to afford (4S-FNL-10) (130 mg,14.3%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.20 (s, 1H), 5.25 (d, J=4.4 Hz, 1H),5.09-4.76 (m, 1H), 4.74-4.68 (m, 1H), 4.60-4.55 (m, 1H), 4.04-3.76 (m,4H), 3.75-3.64 (m, 1H), 3.40-3.31 (m, 1H), 2.02 (s, 3H), 1.93-1.89 (m,2H), 1.86-1.77 (m, 2H), 1.18-1.15 (m, 3H)

LCMS m/z: 352.3 [M⁺+1]

HPLC: 90.18%

Synthesis of tert-butyl2-(hydroxymethyl)-2-((pyrimidin-2-ylmethyl)carbamoyl)pyrrolidine-1-carboxylate(4S-14)

To a stirring solution of compound 4S-W (5 g, 20.4 mmol) in CH₂Cl₂ (50mL) were added DIPEA (11 mL, 61.2 mmol), EDCI (5.84 g, 30.6 mmol), HOBT(4.68 g, 30.6 mmol), 4S-AP (3.52 g, 24.4 mmol) at 0° C. and stirred toRT for 12 h. After consumption of the starting material (by TLC), thereaction mixture was diluted with water (50 mL) and extracted withCH₂Cl₂ (2×100 mL). The combined organic layer was washed with citricacid (1×100 mL) followed by bicarbonate solution (1×100 mL). The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. Obtained crude material was purified by silica gelcolumn chromatography eluting with 2% MeOH/DCM to afford compound 4S-14(2.5 g, 36.5%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.75 (d, J=4.8 Hz, 2H), 8.23 (d, J=5.2 Hz,1H), 7.38 (t, J=5.2 Hz, 1H), 5.16 (t, J=5.2 Hz, 1H), 4.53-4.41 (m, 2H),3.92-3.82 (m, 2H), 3.67-3.63 (m, 1H), 3.54-3.48 (m, 1H), 2.32-2.19 (m,2H), 2.15-1.97 (m, 2H), 1.39 (s, 9H).

Mass (ESI): m/z 337.4 [M⁺+1].

Synthesis of tert-butyl 1-oxo-2-(pyrimidin-2-ylmethyl)-2,5-diazaspiro[3.4] octane-5-carboxylate (4S-15)

To a stirring solution of triphenylphosphine (4.87 g, 18.6 mmol) in THF(30 mL) was added DIAD (3.0 g, 14.88 mmol) at RT and stirred for 30 min.After added compound 4S-14 (2.5 g, 7.44 mmol) in (30 mL) THF slowly andreaction mixture was stirred at RT for 8 h. After consumption of thestarting material (by TLC), the reaction was concentrated under reducedpressure. The crude material was purified by silica gel columnchromatography eluting 80% EtOAc/n-hexane to afford compound 4S-15 (1.2g, 52%) as an off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.80 (d, J=5.2 Hz, 2H), 7.44 (t, J=4.8 Hz,1H), 4.78-4.74 (m, 1H), 4.35-4.31 (m,1H), 3.69-3.60 (m, 1H), 3.42-3.33(m, 2H), 3.28-3.23 (m, 1H), 2.18-2.14 (m, 2H), 1.90-1.76 (m, 2H), 1.40(s, 9H).

Mass (ESI): m/z 319.3 [M⁺+1].

Synthesis of 2-(pyrimidin-2-ylmethyl)-2,5-diazaspiro [3.4] octan-1-one(4S-16)

To a stirring solution of compound 4S-15 (1.5 g, 4.71 mmol) in CH₂Cl₂(20 mL) was added TFA (1.5 mL, 18.86 mmol) at 0° C. and stirred at RTfor 2 h. After completion of reaction (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford 4S-16 (1.4 g, 93%) asreddish syrup (TFA salt).

¹H-NMR: (400 MHz, D₂O): δ 8.84 (d, J=5.2 Hz, 2H), 7.56 (t, J=4.8 Hz,1H), 4.88-4.73 (m, 2H), 3.97 (d, J=6.8 Hz, 1H), 3.82 (d, J=7.2 Hz, 1H),3.55-3.51 (m, 2H), 2.57-2.40 (m, 2H), 2.28-2.15 (m, 2H).

LCMS (M/Z) m/z: 219.3 [M⁺+1].

HPLC: 94.4% (both isomers).

Synthesis of tert-butyl((2S,3R)-3-hydroxy-1-oxo-1-(1-oxo-2-(pyrimidin-2-ylmethyl)-2,5-diazaspiro[3.4] octan-5-yl)butan-2-yl)carbamate (4S-FNL-12)

To a stirring solution of compound 4S-16 (200 mg, 0.63 mmol) in DMF (2mL) were added DIPEA (0.33 mL, 1.89 mmol), 4S-B (160 mg, 0.75 mmol)followed by HATU (289 mg, 0.75 mmol), at 0° C. and stirred for 12 h atRT. After consumption of the starting material (by TLC), the reactionmixture was evaporated under reduced pressure to Obtained crude materialwhich was purified by silica gel column chromatography eluting 1%MeOH/DCM to afford (4S-FNL-12) (70 mg, 26.5%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.79 (d, J=4.8 Hz, 2H), 7.43 (t, J=4.8 Hz,1H), 6.65 (d, J =6.0 Hz, 1H), 6.36 (d, J=8.8 Hz, 1H), 4.80 (s,2H),4.30-4.22 (m, 2H), 4.06-3.83 (m, 2H), 3.73-3.57 (m, 1H), 3.36-3.34 (m,1H), 2.17-2.08 (m, 2H), 1.90-1.88 (m, 2H), 1.38 (s, 9H), 1.06 (d, J=6.8Hz, 3H).

Mass (ESI): m/z 420.5 [M⁺+1].

HPLC: 95.8% (both isomers).

Synthesis of5-((2S,3R)-3-hydroxy-2-((2,2,2-trifluoroacetyl)-14-azanyl)butanoyl)-2-(pyrimidin-2-ylmethyl)-2,5-diazaspiro[3.4] octan-1-one (4S-17)

To a stirring solution of compound (4S-FNL-12) (500 mg, 1.19 mmol) inCH₂Cl₂ (5 mL) was added TFA (680 mg, 5.96 mmol) at 0° C. and stirred atRT for 4 h. After completion of reaction (by TLC), the reaction mixturewas concentrated under reduced pressure to afford crude compound wastriturated with 50% EtOAc/n-hexane (10 mL) to obtained compound 4S-17(400 mg, 77%) as sticky solid (TFA salt).

¹H-NMR: (500 MHz, D₂O): δ 8.47 (d, J=9.0 Hz, 1H), 7.71 (d, J=8.5 Hz,2H), 5.00-4.95 (m, 1H), 4.64-4.59 (m, 1H), 3.90-3.85 (m, 1H), 3.77-3.71(m, 1H), 3.61-3.55 (m, 2H), 3.25-3.20 (m, 2H), 2.41-2.21 (m, 2H),2.15-2.05 (m, 2H), 1.32-1.26 (m, 3H)

Synthesis ofN-((2S,3R)-3-hydroxy-1-oxo-1-(1-oxo-2-(pyrimidin-2-ylmethyl)-2,5-diazaspiro[3.4] octan-5-yl)butan-2-yl)acetamide (4S-FNL-13)

To a stirring solution of compound 4S-17 (500 mg, 1.56 mmol) inCH₂Cl₂/H₂O (5 mL/5 mL) were added NaHCO₃ (658 mg, 7.83 mmol), acetylchloride (367 mg, 4.68 mmol) at 0° C. and stirred at RT for 16 h.Starting material was not completely consumed (observed by TLC). Thereaction mixture was extracted by (2×20 mL) of 10% MeOH/DCM. Thecombined organic layers were dried over Na₂SO₄ and concentrated underreduced pressure to afford crude compound which was purified by silicagel column chromatography eluting 7% MeOH/DCM followed by preparativeHPLC purification to afford (4S-FNL-13) (25 mg, 4.4%) as yellow solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.79 (d, J=5.2 Hz, 2H), 8.03 (d, J=7.6 Hz,1H), 7.43 (t, J =4.8 Hz, 1H), 4.81-4.75 (m, 2H), 4.59-4.56 (m, 1H),4.35-4.25 (m, 1H), 3.89-3.81 (m, 2H), 3.77-3.66 (m, 1H), 3.64-3.56 (m,2H), 2.20-2.11 (m, 2H), 2.08-2.05 (m, 2H), 1.86 (s, 3H), 1.07-1.03 (m,3H)

LCMS (m/z): 362.41 [M⁺+1]

HPLC: 90%

Synthesis of 6-methyl-2,5-diazaspiro [3.4] octan-1-one (4S-18)

To a stirring solution of compound 4S-AE (3 g, 7.79 mmol) in methanol(20 mL) was added 10% Pd/C (1.2 g) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 2h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of ciliate and washed with methanol.Obtained filtrate was concentrated under reduced pressure to affordcompound 4S-18 as yellow solid (1.5 g, 78%).

¹H-NMR: (500 MHz, DMSO-d₆): δ4.15-4.12 (m, 1H), 3.76-3.70 (m, 2H),3.18-3.14 (m, 1H), 3.06 (d, J=4.8 Hz, 1H), 1.33-1.28 (m, 4H), 1.18 (d,J=3.2 Hz, 3H).

LCMS (ESI): 141.2 [M⁺+1].

Synthesis of (4S,5R)-benzyl2,2,5-trimethyl-4-(6-methyl-1-oxo-2,5-diazaspiro [3.4]octane-5-carbonyl)oxazolidine-3-carboxylate (4S-19)

To a stirring solution of 4S-18 (6.3 g, 20.2 mmol) in DCM (30 mL) wasadded compound 4S-18 (2.58 g, 18.42 mmol), TEA (6.26 g, 61.43 mmol) at0° C. under N₂ atmosphere. The reaction mixture was stirred at RT for 2h. After consumption of the starting material (by TLC), the reactionmixture was diluted with water (25 mL).The separated organic layer wasdried over anhydrous Na₂SO₄ and concentrated under reduced pressure.Obtained crude material was purified by column chromatography by using2.5% MeOH/DCM to afford compound 4S-19 as green solid (1.5 g, 20%).

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.79 (s, 1H), 7.40-7.28 (m, 5H), 5.09-4.95(m, 1H), 4.89-4.75 (m, 1H), 4.26-3.91 (m, 2H), 3.80-3.37 (m, 2H),2.05-1.96 (m, 2H), 1.73-1.62 (m, 3H), 1.50 (s, 6H), 1.24-1.14 (m, 3H),1.07-0.85 (m, 3H).

Synthesis of benzyl ((2S,3R)-3-hydroxy-1-(6-methyl-1-oxo-2,5-diazaspiro[3.4] octan-5-yl)-1-oxobutan-2-yl)carbamate (4S-20)

To a stirring solution of compound 4S-19 (1.5 g, 3.61 mmol) in water (30mL) was added TFA (15 mL) at 0° C. and reaction mixture was stirred atRT for 4 h. After consumption of the starting material (by TLC) reactionmixture was extracted with EtOAc (2×30 mL). Combined organic layer andwashed with aqueous NaHCO₃ solution, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to obtained crude material waspurified by column chromatography by using 5% MeOH/DCM to affordcompound 4S-20 (350 mg, 26%) as off-white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.81 (s, 1H), 7.55-7.34 (m, 5H), 4.97 (s,2H), 4.76 (d, J=6.4 Hz, 6.4 Hz, 1H), 4.41-4.35 (m, 1H), 4.14-4.12 (m,1H), 3.79-3.72 (m, 2H), 3.59 (d, J=3.2 Hz, 1H), 2.99 (d, J=4.4 Hz, 4.0Hz, 1H), 2.24-2.16 (m, 1H), 2.06-1.88 (m, 2H), 1.63 (t, J=5.2 Hz, 1H),1.22 (d, J=6.8 Hz, 3H), 1.10 (d, J=6.8 Hz, 3H).

LCMS m/z: 376.4 [M⁺+1].

Synthesis of 6-methyl-2-(2-oxo-2-(pyrrolidin-1-yl) ethyl)-2,5-diazaspiro[3.4] octan-1-one (4S-FNL-14)

To a stirring solution of compound 4S-20 (250 mg, 0.66 mmol) in EtOAc(10 mL) was added 10% Pd/C (100 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 8h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and washed with EtOAc (10mL). Obtained filtrate was concentrated under reduced pressure to afford(4S-FNL-14) as yellow syrup (80 mg, 50%).

¹H-NMR : (400 MHz, DMSO-d₆): δ 7.98 (br s, 1H), 7.49 (br s, 1H), 4.70(d, J=7.2 Hz, 1H), 4.07-4.00 (m, 2H), 3.62 (t, J=3.2 Hz, 1H), 2.92 (s,1H), 2.77-2.69 (m, 2H), 2.14-2.03 (m, 1H), 1.95-1.83 (m, 2H), 1.47-1.40(m, 1H), 1.23 (d, J=6.4 Hz, 3H), 1.11 (d, J=6.4 Hz, 3H).

LCMS m/z: 242.4 [M⁺+1] 92.6%.

Synthesis of benzyl 6-methyl-1-oxo-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro [3.4] octane-5-carboxylate (4S-21)

To a stirring solution of compound 4S-AE (4 g, 14.59 mmol) inacetonitrile (20 mL) was added 4S-AR (2.59 g, 17 mmol), cesiumcarbonate(11.86 g, 36.49 mmol) at 0° C. under N₂ atmosphere. Thereaction mixture was stirred at RT for 2 h. After consumption of thestarting material (by TLC), reaction mixture was filtered through a padof celite and washed with acetonitrile. Obtained filtrate wasconcentrated under reduced pressure to afford crude compound waspurified by column chromatography by eluting 2% MeOH/DCM to affordcompound 4S-21 (3.0 g, 40%) as yellow liquid.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.38-7.31 (m, 5H), 5.11-4.97 (m, 2H), 4.26(s, 1H), 4.01-3.97 (m, 2H), 3.79-3.71 (m, 1H), 3.46-3.36 (m, 2H),3.30-3.22 (m, 2H), 2.30-2.17 (m, 2H), 2.11-2.07 (m, 1H), 2.01-1.97 (m,3H), 1.89-1.84 (m, 2H), 1.79-1.73 (m, 1H), 1.22-1.11 (m, 3H).

LCMS (ESI): 386 [M⁺+1].

Synthesis of 6-methyl-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro[3.4] octan-1-one (4S-22)

To a stirring solution of compound 4S-21 (3 g, 7.79 mmol) in methanol(20 mL) was added 10% Pd/C (1.2 g) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 2h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol. Obtained filtrate was concentrated under reduced pressure toafford compound 4S-22 as yellow solid (1.5 g, 78%).

¹H-NMR: (500 MHz, DMSO-d₆):δ4.01-3.96 (m, 1H), 3.91-3.72 (m, 1H),3.38-3.18 (m, 8H), 2.01-1.83 (m, 6H), 1.77-1.72 (m, 2H), 1.02 (d, J=6.0Hz, 3H).

LCMS (ESI): 251 [M⁺+1].

Synthesis of (4S,5R)-benzyl2,2,5-trimethyl-4-(6-methyl-1-oxo-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro[3.4] octane-5-carbonyl)oxazolidine-3-carboxylate (4S-23)

To a stirring solution of 4S-F (0.6 g, 1.92 mmol) in DCM (10 mL) wasadded to compound 4S-22 (0.48 g, 1.92 mmol), TEA (0.58 g, 5.76 mmol) at0° C. under N₂ atmosphere. Reaction mixture was stirred at RT for 1 h.After consumption of the starting material (by TLC), Reaction mixturewas diluted with water (5 mL).The separated organic layer was dried overanhydrous Na₂SO₄ and concentrated under reduced pressure to obtainedcrude material was purified by column chromatography by using 3%MeOH/DCM to afford compound 4S-23 as brown solid (0.60 g, 56%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.38-7.34 (m, 5H), 5.09-4.74 (m, 2H),4.30-3.92 (m, 4H), 3.85-3.64 (m, 2H), 3.46-3.38 (m, 3H), 2.22-2.01 (m,3H), 1.88-1.74 (m, 5H), 1.58-1.50 (m, 6H), 1.35-1.20 (m, 5H), 1.12-1.07(m, 1H), 0.87-0.78 (m, 2H).

LCMS (ESI): 527 [M⁺+1].

Synthesis of benzyl((2S,3R)-3-hydroxy-1-(6-methyl-1-oxo-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro[3.4] octan-5-yl)-1-oxobutan-2-yl)carbamate (4S-24)

To a stirring solution of compound 4S-23 (0.8 g, 1.52 mmol) in water (5mL) was added TFA (5mL) at 0° C., Reaction mixture was stirred at RT for4 h. After consumption of the starting material (by TLC). Reactionmixture was extracted with EtOAc (2×20 mL). Separated organic layer andwashed with aqueous NaHCO₃ solution, dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to obtained crude material waspurified by column chromatography by using 2% MeOH/DCM to affordcompound 4S-24 (0.25 g, 34%) as yellow solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.56 (d,J=7.6 Hz, 1H), 7.37-7.30 (m, 5H),5.08-4.97 (m, 2H), 4.67 (d, J=6.0 Hz, 1H), 4.52 (t,J=6.4 Hz, 1H),4.42-4.36 (m, 1H), 4.24-4.18 (m, 1H), 4.07 (t,J=8.0 Hz, 1H), 3.80-3.56(m, 4H), 3.38 (t, J=6.8 Hz, 2H), 3.30 (s, 2H), 2.25-2.20 (m, 1H),2.11-2.04 (m, 2H), 1.90-1.82 (m, 2H), 1.78-1.71 (m, 1H), 1.67-1.63 (m,1H), 1.24 (t, J=6.8 Hz, 3H), 1.09-1.02 (m, 3H).

LCMS m/z: 487.4 [M⁺+1].

Synthesis of5-((2S,3R)-2-Amino-3-hydroxybutanoyl)-6-methyl-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro[3.4] octan-1-one (4S-FNL-15)

To a stirring solution of compound 4S-24 (0.15 g, 0.308 mmol) inmethanol (5 mL) was added 10% Pd/C (0.06 g) under N₂ atmosphere.Reaction mixture was stirred under H₂ atmosphere (balloon pressure) for6 h at RT. After consumption of the starting material (by TLC), reactionmixture was filtered through a pad of celite and washed with methanoland concentrated under reduced pressure to afford (4S-FNL-15) as yellowsolid (0.075g, 69%).

¹H-NMR: (400 MHz, D₂O): δ4.52-4.46 (m, 3H), 4.12-3.81 (m, 1H), 3.59-3.52(m, 1H), 3.47-3.43 (m, 5H), 3.06-2.87 (m, 1H), 2.35-2.12 (m, 8H), 1.30(s, 6H).

LCMS (ESI): 353.2 [M⁺+1];

HPLC: 99.72%

Synthesis of 3-methyl-2,5-diazaspiro [3.4] octan-1-one (4S-25)

To a stirring solution of compound 4S-AI (2.50 g, 9.12 mmol) in EtOAc(50 mL) was added 10% Pd/C (500 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 4h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withEtOAc (30 mL). Obtained filtrate was concentrated under reduced pressureto afford crude compound which was purified by column chromatography byeluting 4% MeOH/DCM to afford compound 4S-25 as pale yellow thick syrup(1.0 g, 78.7%).

¹H-NMR: (500 MHz, DMSO-d₆): δ7.79(br s, 1H), 3.39-3.35 (m, 1H),2.92-2.85 (m, 2H), 2.80-2.49 (m, 1H), 1.75-1.64 (m, 2H), 1.62-1.60 (m,2H), 1.10-1.06 (m, 3H).

LCMS (ESI): 141.3 [M⁺+1].

Synthesis of benzyl ((2S,3R)-3-hydroxy-1-(1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-5-yl)-1-oxobutan-2-yl)carbamate (4S-26)

To a stirring solution of compound 4S-25 (1.0 g, 7.13 mmol) in DMF (20mL) were added N, N-diisopropylethylamine (3.94 mL, 21.3 mmol), 4S-A(2.70 g, 10.67 mmol), followed by HATU (5.42 g, 14.26 mmol), at 0° C.and stirred at RT for 16 h. After consumption of the starting material(by TLC), the reaction mixture was diluted with water (30 mL) and EtOAc(50 mL). The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to obtained crude compound which waspurified by column chromatography eluting 2% MeOH/DCM to afford compound4S-26 (500 mg, 18.7%) as colorless syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ7.95 (br s, 1H), 7.37-7.28 (m, 5H), 7.05(d, J=8.4 Hz, 1H) 5.08-4.98 (m, 2H), 4.78-4.71 (m, 1H), 4.33-4.11 (m,1H), 3.99-3.72 (m, 2H), 3.68-3.44 (m, 2H), 1.97-1.74 (m, 4H), 1.12-1.11(m, 3H), 1.06 (t, J=6.4 Hz, 3H).

Synthesis of5-((2S,3R)-2-amino-3-hydroxybutanoyl)-3-methyl-2,5-diazaspiro [3.4]octan-1-one (4S-FNL-16)

To a stirring solution of compound 4S-26 (500 mg, 1.33 mmol) in EtOAc(20 mL) was added 10% Pd/C (100 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 8h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withEtOAc (20 mL). Obtained filtrate was concentrated under reduced pressureto afford crude compound which was purified by column chromatography byeluting 2% MeOH/DCM to afford (4S-FNL-16) as pale yellow thick syrup(170 mg, 52.8%).

¹H-NMR: (400 MHz, DMSO-d₆): δ7.42 (s, 1H), 4.68 (d, J=6.4 Hz, 1H),4.10-4.00 (m, 1H) 3.79-3.76 (m, 1H), 3.38-3.33 (m, 1H), 3.04-2.97 (m,1H), 3.38-3.33 (m, 1H), 3.04-2.97 (m, 1H), 2.16-2.14 (m, 1H), 1.97-1.64(m, 4H), 1.20-1.04 (m, 6H).

LCMS (ESI): 242.1 [M⁺+1].

Synthesis of benzyl1-methyl-3-oxo-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro [3.41]octane-5-carboxylate (4S-27)

To a stirring solution of compound 4S-AI (500 mg, 1.82 mmol) inacetonitrile (10 mL) were added 4S-AR (377 mg, 2.55 mmol), cesiumcarbonate(1.48 g, 4.56 mmol) at 0° C. under N₂ atmosphere. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was diluted with water (20 mL)and extracted with EtOAc (30 mL). The separated organic layer was driedover Na₂SO₄, concentrated under reduced pressure to afford crudecompound, which was purified by column chromatography eluting 2%MeOH/DCM to afford compound 4S-27 (400 mg, 56.8%) as yellow liquid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.39-7.31 (m, 5H), 5.08-5.04 (m, 2H),4.11-3.95 (m, 2H), 3.87 (s, 1H), 3.60-3.47 (m, 4H), 2.18-2.14 (m, 2H),1.86-1.74 (m, 7H), 1.23-1.11 (m, 1H), 1.06 (d, J=6.4 Hz, 3H).

LCMS (ESI): 386.4 [M⁺+1].

Synthesis of 3-methyl-2-(2-oxo-2-(pyrrolidin-1-yl) ethyl)-2,5-diazaspiro[3.4] octan-1-one 4S-28:

To a stirring solution of compound 4S-7 (400 mg, 1.03 mmol) in methanol(10 mL) was added 10% Pd/C (80 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 4h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol (10 mL). Obtained filtrate was concentrated under reducedpressure to afford crude compound was purified by column chromatographyby eluting 2% MeOH/DCM to afford 4S-28 as pale yellow thick syrup (160mg, 61.3%).

¹H-NMR: (400 MHz, DMSO-d₆): δ4.06-3.99 (m, 1H), 3.77-3.70 (m, 1H),3.63-3.58 (m, 1H), 3.39 (s, 2H), 3.27 (t, J=7.2 Hz, 2H), 2.94-2.76 (m,3H), 1.98-1.59 (m, 8H), 1.06 (d, J=6.4 Hz, 3H).

LCMS (ESI): 252.3 [M⁺+1].

ELSD: 32.06 and 67.69%.

Synthesis of benzyl(3-hydroxy-1-(1-methyl-3-oxo-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro [3.4] octan-5-yl)-1-oxobutan-2-yl)carbamate(4S-29)

To a stirring solution of 4S-28 (600 mg, 2.38 mmol) in CH₂Cl₂ (20 mL)were added N, N-diisopropylethylamine (1.31 mL, 7.16 mmol), 4S-A(Cbz-threonine, 906 mg, 3.58 mmol), HATU (1.81 g, 4.77 mmol), at 0° C.and stirring was continued at RT for 16 h. The reaction mixture wastreated with saturated aqueous NaHCO₃ solution for 15 min. The aqueouslayer was extracted with CH₂Cl₂ (2×20 mL), the organic layer wasseparated and washed with saturated citric acid solution. The organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure to obtained crude product, which was eluted with 2%MeOH/DCM to afford compound 4S-29 (280 mg, 24%) as pale yellow syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 7.37-7.27 (m,SH), 7.04 (s, 1H), 5.07-4.98(m, 2H), 4.77-4.70 (m, 1H), 4.29-4.26 (m, 1H), 4.15-3.96 (m, 2H),3.86-3.56 (m, 4H), 3.48-3.35 (m, 2H), 3.30-3.25 (m, 2H), 2.13-2.04 (m,1H), 1.88-1.74 (m, 7H), 1.14 (d, J=6.4 Hz, 3H), 1.07-1.05 (m, 3H). LCMS(ESI): 487.6 [M⁺+1]

Synthesis of5-(2-amino-3-hydroxybutanoyl)-3-methyl-2-(2-oxo-2-(pyrrolidin-1-yl)ethyl)-2,5-diazaspiro [3.4] octan-1-one (4S-FNL-18)

To a stirring solution of compound 4S-29 (140 mg, 0.28 mmol) in methanol(10 mL) was added 10% Pd/C (80 mg) under N₂ atmosphere. The reactionmixture was stirred under H₂ atmosphere (balloon pressure) at RT for 3h. After consumption of the starting material (by TLC), the reactionmixture was filtered through a pad of celite and the pad was washed withmethanol (10 mL). Obtained filtrate was concentrated under reducedpressure to afford crude compound was purified by column chromatographyby eluting 4% MeOH/DCM to afford compound (4S-FNL-18) as off-whitesticky solid (160 mg, 61.3%).

¹H-NMR: (400 MHz, DMSO-d₆): δ 5.27 (d, J=4.4 Hz, 1H), 4.71-4.66 (m, 1H),4.13-4.05 (m, 2H), 3.65-3.55 (m, 1H), 3.27-3.18 (m, 7H), 2.22-2.18 (m,1H), 1.97-1.71 (m, 8H), 1.16-0.98 (m, 6H).

LCMS (ESI): 353.3 [M⁺+1].

Synthesis of tert-butyl2-(2-amino-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro [3.4]octane-5-carboxylate (4S-30)

To a stirring solution of 4S-AO (2 g, 6.13 mmol) in ethanol (5 mL) wasadded ethanolic ammonia (20 mL) at 0° C. in sealed tube. The reactionmixture was stirred at RT for 16 h. After consumption of the startingmaterial (by TLC), the reaction mixture was concentrated under reducedpressure to afford compound 4S-30 (1.5 g, 83%) as white sticky solidused directly for next step without purification.

¹H-NMR: (400 MHz, DMSO-d₆) δ 7.52 (s, 2H), 3.98-3.81 (m, 1H), 3.73-3.52(m, 2H), 3.49-3.31 (m, 2H), 2.12-1.70 (m, 4H), 1.40 (s, 9H), 1.18 (d,J=6.4 Hz, 3H);

LCMS (ESI): m/z 298.3 [M⁺+1]

Synthesis of 2-(1-methyl-3-oxo-2,5-diazaspiro [3.4] octan-2-yl)acetamide(4S-31)

To a stirring solution of compound 4S-30 (1.5 g, 5.05 mmol) in DCM (40mL) was added TFA (3.85 mL, 50.5 mol) slowly and stirred to RT for 3 h.After consumption of the starting material (by TLC), the reaction wasconcentrated under reduced pressure to afford compound 4S-31 (1.5 g,crude, TFA salt) was used directly for next step without anypurification.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.45 (s, 2H), 7.49 (d, J=15.5 Hz, 1H),3.99-3.40 (m, 3H), 3.39-3.26 (m, 2H), 2.27-1.90 (m, 4H), 1.17-1.11 (m,3H);

LCMS (ESI): m/z 198.2 [M⁺+1]

Synthesis of tert-butyl(4S,5R)-4-(2-(2-amino-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro [3.4]octane-5-carbonyl)-2,2,5-trimethyloxazolidine-3-carboxylate (4S-32)

To a stirring solution of compound 4S-31 (1.5 g, 4.82 mmol) in DMF (20mL) were added N,N-diisopropylethylamine (4.19 mL, 24.1 mmol), 4S-G(1.62 g, 6.26 mmol) followed by HATU (2.74 g, 7.23 mmol) at 0° C. andstirred at RT for 16 h. After consumption of the starting material (byTLC), the residue was diluted with water (100 mL) and EtOAc (100 mL).The separated organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure to afford crude compound which waspurified by column chromatography by eluting 2% MeOH/DCM to affordcompound 4S-32 (1.2 g) as brown thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.61 (s, 2H), 4.26-4.06 (m, 1H), 4.00-3.93(m, 2H), 3.92-3.74 (m, 2H), 3.63-3.43 (m, 2H), 2.36-1.99 (m, 1H),1.92-1.67 (m, 3H), 1.52 (s, 6H), 1.48-1.39 (m, 3H), 1.39 (s, 9H),1.20-1.17 (m, 3H);

LCMS (ESI): m/z 439.5 [M⁺+1]

Synthesis of tert-butyl (4S,5R)-4-(2-(2-(((E)-(dimethylamino)methylene)amino)-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro [3.4]octane-5-carbonyl)-2,2,5-trimethyloxazolidine-3-carboxylate (4S-33)

To a stirring solution of compound 4S-32 (2.3 g, 5.24 mmol) in DMF.DMA(11.5 mL) was heated to 80° C. for 4 h. After consumption of thestarting material (by TLC), the reaction mixture was concentrated underreduced pressure to afford compound 4S-33 (2.6 g, crude) as brown thicksyrup was used directly for next step without purification.

Synthesis of tert-butyl(4S,5R)-4-(2-((1,2,4-oxadiazol-5-yl)methyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octane-5-carbonyl)-2,2,5-trimethyloxazolidine-3-carboxylate(4S-34)

To a stirring solution of compound 4S-33 (2.6 g, 5.26 mmol) in ethanolwas added hydroxyl amine.HCl (732 mg, 10.53 mmol) and heated to 80° C.for 6 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford crudecompound, which was diluted with H₂O (30 mL) and EtOAc (50 mL). Theseparated organic layer was dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure to afford crude residue which was purified bycolumn chromatography by eluting 1% MeOH/DCM to afford compound 4S-34(620 mg, 25.4%) as yellow thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 9.00 (s, 1H), 4.95-4.62 (m, 2H), 4.35-4.01(m, 1H), 3.94-3.42 (m, 2H), 2.89-2.63 (m, 2H), 2.17-1.86 (m, 4H), 1.51(s, 6H), 1.41 (s, 9H), 1.28-1.10 (m, 6H)

LCMS: m/z 464.5 [M⁺+1]

Synthesis of tert-butyl((2S,3R)-1-(2-((1,2,4-oxadiazol-5-yl)methyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl)carbamate (4S-FNL-19)

To a stirring solution of compound 4S-34 (620 mg, 1.33 mmol) in methanol(10 mL) was added PTSA (254 mg, 1.33 mmol) at RT and stirred for 16 h.After consumption of the starting material (by TLC), the reactionmixture was quenched with NaHCO₃ and stirred for 15 minutes. Afterfiltered the reaction mass and methanol was concentrated under reducedpressure to afford crude compound, which was purified by columnchromatography by eluting 1% MeOH/DCM to afford (4S-FNL-19) (240 mg,42.6%) as an off-white semi solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 8.98 (s, 1H), 6.77 (d, J=7.6 Hz, 1H),4.70-4.62 (m, 2H), 4.23-4.20 (m, 1H), 4.08-4.01 (m, 2H), 3.72-3.55 (m,3H), 2.15-2.06 (m, 1H), 1.90-1.81 (m, 3H), 1.38 (s, 9H), 1.16-1.04 (m,6H);

HPLC:95.96%

LCMS (ESI): m/z 424.4 [M⁺+1]

Synthesis of ethyl 2-(1-methyl-3-oxo-2,5-diazaspiro [3.4]octan-2-yl)acetate (4S-35)

To a stirring solution of 45-AO (2 g, 6.13 mmol) in DCM (20 mL) wasadded trifluoroacetic acid (4.71 mL, 61.2 mmol) at 0° C. under N₂atmosphere. The reaction mixture was stirred at RT for 1 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasconcentrated under reduced pressure to afford crude compound which wastriturated with n-pentane (10 mL) to afford compound 4S-35 (2.08 g,crude) as brown thick syrup (TFA salt).

¹H-NMR: (500 MHz, DMSO-d₆) δ 9.20 (s, 1H), 4.22-3.98 (m, 3H), 3.45-3.33(m, 2H), 3.31-3.26 (m, 2H), 2.23-2.05 (m, 3H), 2.04-1.50 (m, 2H),1.44-1.31 (m, 2H), 1.30-1.22 (m, 3H)

LCMS m/z: 227.3 [M⁺+1]

Synthesis of ethyl2-(5-(N-((benzyloxy)carbonyl)-O-(tert-butyldimethylsilyl)-L-threonyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-2-yl)acetate (4S-36)

To a stirring solution of compound 4S-35 (2 g, 5.87 mmol) in DMF (10 mL)were added N,N-diisopropylethylamine (2.55 mL, 14.6 mmol), 4S-K (2.58 g,7.03) followed by HATU (3.34 g, 8.8 mmol) at 0° C. and stirred at RT for16 h. After consumption of the starting material (by TLC), the residuewas diluted with water (100 mL) and EtOAc (100 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to obtained crude; which was purified by columnchromatography eluting 50% EtOAc/n-hexane to afford compound 4S-36 (1 g,29.6%) as yellow thick syrup.

¹H-NMR: (500 MHz, DMSO-d₆): δ 7.37-7.31 (m, 5H), 7.19 (d, J=9.5 Hz, 1H),5.01 (s, 2H), 4.35-4.10 (m, 2H), 4.09-4.00 (m, 2H), 3.99-3.64 (m, 4H),2.71-2.60 (m, 4H), 2.10-1.78 (m, 4H), 1.23-1.12 (m, 6H), 1.10 (s, 9H),0.04 (s, 6H)

LCMS m/z: 576.7 [M⁺+1]

Synthesis of benzyl((2S,3R)-3-((tert-butyldimethylsilyl)oxy)-1-(2-(2-hydrazinyl-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-5-yl)-1-oxobutan-2-yl) carbamate (4S-37)

To a stirring solution of compound 4S-36 (1 g, 1.73 mmol) in ethanol (15mL) was added hydrazine hydrate(0.25 mL, 5.21 mmol) and heated to 80° C.for 3 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford crudecompound which was purified by column chromatography by eluting 2%MeOH/DCM to afford compound 4S-37 (850 mg, 87.6%) as an off-white semisolid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.10 (s, 1H), 7.50 (s, 1H), 7.36-7.26 (m,5H), 4.99 (s, 2H), 4.42-4.38 (m, 3H), 4.03-3.92 (m, 2H), 3.87-3.81 (m,1H), 3.70-3.62 (m, 3H), 2.13-2.09 (m, 1H), 1.98-1.74 (m, 3H), 1.23-1.10(m, 6H), 0.81 (s, 9H), 0.05 (s, 6H)

LCMS (ESI): 562.7 [M⁺+1]

Synthesis of benzyl((2S,3R)-3-((tert-butyldimethylsilyl)oxy)-1-(2-(2-hydrazinyl-2-oxoethyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-5-yl)-1-oxobutan-2-yl)carbamate (4S-38)

To a stirring solution of compound 4S-37 (850 mg, 1.51 mmol) in triethylorthoformate (8.5 mL) was added PTSA (28.7 mg, 0.15 mmol) and heated to80° C. for 6 h. After consumption of the starting material (by TLC), thereaction mixture was concentrated under reduced pressure to afford crudecompound, which was purified by column chromatography by eluting 1%MeOH/DCM to afford compound 4S-38 (650 mg, 75.4%) as yellow thick syrup.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.24 (s, 1H), 7.35-7.30 (m, 5H), 5.01 (s,2H), 4.64-4.58 (m, 2H), 4.22-3.89 (m, 4H), 3.67-3.60 (m, 2H), 2.12-2.07(m, 1H), 2.01-1.78 (m, 3H), 1.33-1.12 (m, 6H), 0.82 (s, 9H), 0.01 (s,6H).

LCMS m/z: 571.3 [M⁺+1].

Synthesis of benzyl((2S,3R)-1-(2-((1,3,4-oxadiazol-2-yl)methyl)-1-methyl-3-oxo-2,5-diazaspiro[3.4] octan-5-yl)-3-hydroxy-1-oxobutan-2-yl)carbamate (4S-39)

To a stirring solution of compound 4S-38 (650 mg, 1.13 mmol) in THF (10mL) was added TBAF (1M in THF) (1.7 mL, 1.7 mmol) at 0° C. under N₂atmosphere. The reaction mixture was stirred at RT for 1 h. Afterconsumption of the starting material (by TLC), the reaction mixture wasdiluted with NH₄Cl solution (20 mL) and EtOAc (30 mL). The separatedorganic layer was dried over anhydrous Na₂SO₄ and concentrated underreduced pressure to obtained crude; which was purified by columnchromatography eluting 2% MeOH/DCM to afford compound 4S-39 (300 mg,58.1%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): δ 9.24 (s, 1H), 7.37-7.30 (m, 5H), 7.07 (t,J=8.8 Hz, 1H), 5.07 (s, 2H), 4.81-4.72 (m, 1H), 4.69-4.61 (m, 1H),4.33-4.25 (m, 2H), 3.99-3.85 (m, 2H), 3.40-3.15 (m, 2H), 2.13-2.05 (m,1H), 1.85-1.80 (m, 3H), 1.13-1.03 (m, 6H).

Synthesis of2-((1,3,4-oxadiazol-2-yl)methyl)-5-(L-threonyl)-3-methyl-2,5-diazaspiro[3.4] octan-1-one (4S-FNL-20)

To a stirring solution of compound 4S-39 (300 mg, 0.65 mmol) in methanol(10 mL) was added 10% wet Pd/C (90 mg) and stirred under H₂ atmosphere(balloon pressure) for 12 h at RT. After consumption of the startingmaterial (by TLC), reaction mixture was filtered through a pad of celiteand triturated with methanol (5 mL). The filtrate was concentrated underreduced pressure to afford crude compound which was purified by columnchromatography by eluting 5% MeOH/DCM to afford (4S-FNL-20) (100 mg,47.1%) as white solid.

¹H-NMR: (400 MHz, DMSO-d₆): 9.14 (s, 1H), 7.99-7.94 (m, 1H), 5.16-5.09(m, 1H), 4.11-4.05 (m, 1H), 3.99-3.74 (m, 3H), 3.55-3.52 (m, 1H),3.36-3.27 (m, 1H), 3.17-2.79 (m, 1H), 2.68-2.64 (m, 1H), 2.33-2.18 (m,1H), 1.96-1.63 (m, 3H), 1.17-1.01 (m, 6H);

LCMS m/z: 324.3 [M⁺+1];

HPLC: 95.52% .

Example 3 [³H] MK-801 Binding Assay

Methods

Assays were conducted as described in Moskal et al. (Moskal, J. R., Kuo,A. G., Weiss, C., Wood, P. L., O'Connor Hanson, A., Kelso, S., Harris,R. B., Disterhoft, J. F., 2005. GLYX-13: a monoclonal antibody-derivedpeptide that acts as an N-methyl-D-aspartate receptor modulator.Neuropharmacology. 49, 1077-87) The potentiation of [³H]MK-801 binding(5 nM; 22.5 Ci/mmol) to well washed rat cortical membranes (200 μg) wasmeasured under non-equilibrium conditions (15 min @ 25° C.) in thepresence of increasing concentrations of test compounds and 50 μMglutamate. Zero levels were determined in the absence of any glycineligand and in the presence of 30 μM 5,7 DCKA. Maximal stimulation wasmeasured in the presence of 1 mM glycine, and 50 μM glutamate waspresent in all samples. The facilitation of [³H]MK-801 binding by testscompounds was calculated by using a 3 parameter log agonist vs. responseequation (Graph pad Prism, USA) and potency (EC₅₀, expressed in pM) andmaximal activity (% maximal stimulation) were calculated for the testcompound.

Results

As shown in Table 2, FIG. 1, the potency and maximal activity forCompound X is 0.3 and 31%.

TABLE 2 Activity Compounds pEC50 (%) X 0.3 31

TABLE 3 Additional Biological Data Unified Unified Unified [3H] MK-Activity Activity Unified Unified Activity 801 Unified Unified Data:LTP, Data: Activity Activity Data: binding Activity Activity SignificantPorsolt Data: Data: Porsolt assay: Data: LTP Data: LTP (S) or Non-Floating Time Porsolt Porsolt Time Post EC50 Augmentation Concentrationsignificant Inhibition Dose Dose, Dose Compound (M) (Percent) (uM) (NS)(Percent) (mg/kg) route (Hours) 4S- 2.43E−13 80 1 NS 70 3 IV 1 FNL-1 4S-5.53E−11 FNL-2

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

What is claimed is:
 1. A compound represented by formula I:

or a stereoisomer, or an N-oxide, and/or a pharmaceutically acceptablesalt thereof, wherein R_(b) is H or halogen; R₁ H or methyl; R₂ is H ormethyl; R₃ is —OH or C₁-C₆alkoxy; R₄ is H or methyl; X is selected fromthe group consisting of: (i) —C₁₋₆alkylene- C(O)-heterocyclyl includingfrom 3 to 6 ring atoms wherein 1, 2, or 3 of the ring atoms areindependently selected from the group consisting of N and O; wherein theheterocyclyl is optionally substituted by one, two or three substituentsindependently selected from the group consisting of halogen, hydroxyl,C₁-C₆alkyl, and C₁-C₆alkoxy; (ii) —C₁₋₆alkylene- heteroaryl includingfrom 5 to 6 ring atoms wherein 1, 2, or 3 of the ring atoms areindependently selected from the group consisting of N and O; wherein theheteroaryl is optionally substituted by one, two or three substituentsindependently selected from the group consisting of halogen, hydroxyl,C₁-C₆alkyl, and C₁-C₆alkoxy; and (iii) branched C₃-C₆ alkyl substitutedwith —C(O)NH₂ on one carbon and —OH on another carbon; and wherein the—NH₂ group attached to the carbon adjacent to —CH(R₃)(R₄) is optionallysubstituted with a substituent selected from —C(O)OR₃₁ and —C(O)R₃₂,wherein: R₃₁ is C₁-C₆ alkyl; and R₃₂ is C₁-C₆.
 2. The compound of claim1, wherein R_(b) is H.
 3. The compound of claim 1, wherein R₁ is H. 4.The compound of claim 1, wherein R₁ is methyl.
 5. The compound of claim1, wherein R₂ is H.
 6. The compound of claim 1, wherein R₂ is methyl. 7.The compound of claim 1, wherein R₄ is H.
 8. The compound of claim 1,wherein R₄ is methyl.
 9. The compound of claim 1, wherein X is—CH₂C(O)-heterocyclyl including 5 ring atoms wherein one ring atom is N.10. The compound of claim 1, wherein X is


11. The compound of claim 1, wherein X is branched C₃ alkyl substitutedwith —C(O)NH₂ on one carbon and —OH on another carbon.
 12. The compoundof claim 1, wherein the —NH₂ group attached to the carbon adjacent to—CH(R₃)(R₄) is substituted with a substituent selected from —C(O)OR₃₁and —C(O)R₃₂, wherein R₃₁ is t-butyl and R₃₂ is methyl or isopropyl. 13.A method of treating neuropathic pain, the method comprising:administering to a patient in need thereof a compound represented byformula I:

or a stereoisomer, an N-oxide, and/or a pharmaceutically acceptable saltthereof, wherein R_(b) is selected from the group consisting of H,halogen, hydroxyl, cyano and C₁-C₆ alkyl; R₁ is H or C₁-C₆ alkyl; R₂ isH or C₁-C₆ alkyl; R₃ is selected from the group consisting of H,C₁-C₆alkyl, —OH, C₁-C₆alkoxy, —OC(O)—C₁-C₆ alkyl, and —OC(O)-phenyl,wherein phenyl is optionally substituted by one, two or threesubstituents independently selected from the group consisting ofhalogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy; R₄ is H or C₁-C₆ alkyl;X is selected from the group consisting of: (i)—C₁₋₆alkylene-C₃₋₆cycloalkyl; (ii) —C₁₋₆alkylene-heterocyclyl includingfrom 3 to 6 ring atoms wherein 1, 2, or 3 of the ring atoms areindependently selected from the group consisting of N, O, and S, whereinheterocyclyl is optionally substituted by one, two or three substituentsindependently selected from the group consisting of halogen, hydroxyl,C₁-C₆alkyl, and C₁-C₆alkoxy; (iii) —C₁₋₆alkylene-C(O)-heterocyclylincluding from 3 to 6 ring atoms wherein 1, 2, or 3 of the ring atomsare independently selected from the group consisting of N, O, and S;wherein heterocyclyl is optionally substituted by one, two or threesubstituents independently selected from the group consisting ofhalogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy; (iv)—C₁₋₆alkylene-heteroaryl including from 5 to 6 ring atoms wherein 1, 2,or 3 of the ring atoms are independently selected from the groupconsisting of N, O, and S; wherein heteroaryl is optionally substitutedby one, two or three substituents independently selected from the groupconsisting of halogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy; and (v)branched C₃-C₆ alkyl substituted with —C(O)NH₂ on one carbon and —OH onanother carbon; and wherein the —NH₂ group attached to the carbonadjacent to —CH(R₃)(R₄) is optionally substituted with a substituentselected from —C(O)OR₃₁ and —C(O)R₃₂, wherein: R₃₁ is selected from thegroup consisting of C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₂-C₆ alkenyl, C₂-C₆alkenyl, C₃-C₁₀ cycloalkyl, —CH₂—C₃-C₁₀ cycloalkyl, —CH₂-phenyl, and—CH₂-pyridyl, wherein C₃-C₁₀ cycloalkyl is optionally substituted withfrom 1-3 independently selected C₁-C₃ alkyl, and phenyl is optionallysubstituted with from 1-2 substituents independently selected from C₁-C₃alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy, C₁-C₃ haloalkoxy, nitro, halo,SO₂Me, cyano, and —OC(O)CH₃; and R₃₂ is selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ haloalkyl, phenyl, and pyridyl,wherein phenyl is optionally substituted with from 1-2 substituentsindependently selected from C₁-C₃ alkyl, C₁-C₃ haloalkyl, C₁-C₃ alkoxy,C₁-C₃ haloalkoxy, nitro, halo, SO₂Me, cyano, and —OC(O)CH₃.
 14. Themethod of claim 13, wherein R_(b) is H; R₁ is H or methyl; R₂ is H ormethyl; R₃ is —OH; and R₄ is H or methyl.
 15. The method of claim 13,wherein X is —C₁₋₂alkylene-C(O)-heterocyclyl including from 3 to 6 ringatoms wherein 1, 2, or 3 of the ring atoms are independently selectedfrom the group consisting of N and O; wherein heterocyclyl is optionallysubstituted by one, two or three substituents independently selectedfrom the group consisting of halogen, hydroxyl, C₁-C₆alkyl, andC₁-C₆alkoxy.
 16. The method of claim 13, wherein X is—C₁₋₂alkylene-heteroaryl including from 5 to 6 ring atoms wherein 1, 2,or 3 of the ring atoms are independently selected from the groupconsisting of N and O; wherein heteroaryl is optionally substituted byone, two or three substituents independently selected from the groupconsisting of halogen, hydroxyl, C₁-C₆alkyl, and C₁-C₆alkoxy.
 17. Themethod of claim 13, wherein X is branched C₃-C₄ alkyl substituted with—C(O)NH₂ on one carbon and —OH on another carbon.
 18. The method ofclaim 13, wherein the neuropathic pain is chronic.
 19. The method ofclaim 13, wherein the neuropathic pain is associated with fibromyalgia.20. The method of claim 13, wherein the neuropathic pain is associatedwith diabetic neuropathy.